Compositions and methods for altering alpha- and beta-tocotrienol content using multiple transgenes

ABSTRACT

Preparation and use of isolated nucleic acids useful in altering the oil phenotype of plants are described. Isolated nucleic acids and their encoded polypeptides are described that alter the content of alpha-tocotrienol, beta-tocotrienol, or both, in transformed seeds and oil obtained from the transformed seeds. Expression cassettes, host cells and transformed plants are described that contain the foregoing nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/523,997, filed Jun. 15, 2012 and issued on Feb. 12, 2013 as U.S.Pat. No. 8,372,623, which is a continuation application of U.S.application Ser. No. 13/005,369, filed Jan. 12, 2011 and issued on Oct.23, 2012 as U.S. Pat. No. 8,293,976, which is a divisional applicationof U.S. application Ser. No. 12/240,434, filed Sep. 29, 2008 and issuedon Feb. 22, 2011 as U.S. Pat. No. 7,893,324, which claims the benefit ofU.S. Provisional Application No. 60/977,495, filed Oct. 4, 2007, theentire disclosure of which is herein incorporated by reference.

FIELD OF INVENTION

The field of the invention relates to plant breeding and molecularbiology, and particularly to alteration of oil phenotype in plantsthrough the use of nucleic acid fragments encoding homogentisategeranylgeranyl transferase, gamma-tocopherol methyltransferase (VTE4)and 2-methyl-6-phytylbenzoquinol methyltransferase (VTE3).

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named400523SEQLIST.txt, created on Jan. 12, 2011, and having a size of 251kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Tocotrienols are vitamin E-related compounds whose occurrence in plantsis limited primarily to the seeds and fruits of most monocot species(e.g., palm, wheat, rice and barley). Tocotrienols are structurallysimilar to tocopherols, including alpha-tocopherol which is a form ofvitamin E. Tocopherols occur ubiquitously in the plant kingdom as wellas in photosynthetic microbes such as Synechocystis.

Tocotrienols and tocopherols both contain a chromanol head group that islinked to a hydrocarbon side chain. The only structural differencebetween these molecules is the presence of three double bonds in thehydrocarbon side chain of tocotrienols. This difference is related tothe biosynthetic origins of the side chains. Tocopherol side chains arederived from phytyl-pyrophosphate (PP), and the tocotrienol side chainsare believed to be derived from geranylgeranyl-PP, see FIG. 1 and FIG.2, respectively (Soll et al. (1980) Arch. Biochem. Biophys.204:544-550).

At least four forms or molecular species of tocopherols and tocotrienolsoccur in nature: alpha, beta, gamma and delta (α, β, γ and δ,respectively). These molecular species contain different numbers ofmethyl groups that are bound to the aromatic portion of the chromanolhead. Like tocopherols, tocotrienols are potent lipid-solubleantioxidants and therefore have considerable nutritive value in humanand animal diets (Packer et al. (2001) J. Nutr. 131:369 S-373S). Inaddition, tocotrienols are believed to have therapeutic propertiesincluding a demonstrated ability to down regulate cholesterolbiosynthesis (Theriault et al. (1999) Clin. Biochem. 32:309-319;Qureshii et al. (1986) J. Biol. Chem. 261:10544-10550).

The first committed step in the tocopherol biosynthetic pathway is theprenylation of homogentisic acid with phytyldiphosphate to form2-methyl-6-phytylbenzoquinol (MPBQ). Two distinct methyltransferaseenzymes catalyze methylations of the aromatic moiety of tocopherols(VTE3 and VTE4). 2-methyl-6-phytylbenzoquinol methyltransferase (VTE3)acts on the tocopherol intermediate MPBQ prior to cyclization.Cyclization of the product of the first methylation reaction(2,3-dimethyl-5-phytylbenzoquinol) with tocopherol cyclase (VTE1)provides gamma-tocopherol. Gamma-tocopherol is further methylated toalpha-tocopherol by the second methyltransferase enzyme of tocopherolbiosynthesis, gamma-tocopherol methyltransferase (VTE4). The same enzymemethylates delta-tocopherol thereby generating beta-tocopherol.

It has been speculated that the first committed step in the biosynthesisof tocotrienols involves the condensation of geranylgeranyl-PP andhomogentisate to form 2-methyl-6-geranylgeranylbenzoquinol (Soll et al.(1980) Arch. Biochem. Biophys. 204:544-550). The enzyme that catalyzesthis reaction can thus be functionally described as a homogentisategeranylgeranyl transferase (HGGT). After cyclization and an initialmethylation, the last step of tocotrienol production would require themethylation of gamma-tocotrienol to alpha-tocotrienol ordelta-tocotrienol to beta-tocotrienol.

Functional identification of genes or cDNAs encoding homogentisategeranylgeranyl transferase (HGGT) and gamma-tocopherol methyltransferasepolypeptides has been reported. The use of these nucleic acids incombination to manipulate the biosynthesis of the nutritionallyimportant tocotrienols, such as alpha- and beta-tocotrienol, in plants,seeds and microbial hosts has been reported in US Patent PublicationUS-2007-0199096-A1.

SUMMARY OF THE INVENTION

Compositions and methods for the alteration of the alpha- andbeta-tocotrienol content and composition of plants are provided. Thecompositions comprise nucleotide molecules comprising nucleotidesequences for HGGT, gamma-tocopherol methyltransferase and2-methyl-6-phytylbenzoquinol methyltransferase. The compositions can beused to transform plants to manipulate the synthetic pathway for tocolcompounds.

The present invention includes:

In one embodiment, a transformed plant comprising in its genome: (a) afirst recombinant nucleic acid molecule comprising at least oneregulatory sequence operably linked to at least one nucleotide sequenceselected from the group consisting of: (i) a nucleotide sequenceencoding a polypeptide having gamma-tocopherol methyltransferaseactivity; (ii) a nucleotide sequence set forth in SEQ ID NOs: 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39; (iii) a nucleotidesequence encoding the amino acid sequence set forth in SEQ ID NOs: 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40; (iv) anucleotide sequence having at least 80% sequence identity to thenucleotide sequence set forth in any one of (i)-(iii), wherein thenucleotide sequence encodes a polypeptide having gamma-tocopherolmethyltransferase activity; and (v) a nucleotide sequence that is fullycomplementary to the nucleotide sequence of any one of (i)-(iv); (b) asecond recombinant nucleic acid molecule comprising at least oneregulatory sequence operably linked to at least one nucleotide sequenceselected from the group consisting of: (vi) a nucleotide sequenceencoding a polypeptide having homogentisate geranylgeranyl transferaseactivity; (vii) a nucleotide sequence set forth in SEQ ID NOs: 1, 3, 5,7, or 9; (viii) a nucleotide sequence encoding the amino acid sequenceset forth in SEQ ID NOs: 2, 4, 6, 8, or 10; (ix) a nucleotide sequencehaving at least 80% sequence identity to the nucleotide sequence setforth in any one of (vi)-(viii), wherein the nucleotide sequence encodesa polypeptide having homogentisate geranylgeranyl transferase activity;and (x) a nucleotide sequence that is fully complementary to thenucleotide sequence of any one of (vi)-(ix); and (c) a third recombinantnucleic acid molecule comprising at least one regulatory sequenceoperably linked to at least one nucleotide sequence selected from thegroup consisting of: (xi) a nucleotide sequence encoding a polypeptidehaving 2-methyl-6-phytylbenzoquinol methyltransferase activity; (xii) anucleotide sequence set forth in SEQ ID NO:53; (xiii) a nucleotidesequence encoding the amino acid sequence set forth in SEQ ID NOs: 54,61, 62, 63, 64, 65, 66, 67, 68, 69 or 70; (xiv) a nucleotide sequencehaving at least 80% sequence identity to the nucleotide sequence setforth in any one of (xi)-(xii), wherein the nucleotide sequence encodesa polypeptide having 2-methyl-6-phytylbenzoquinol methyltransferaseactivity; (xv) a nucleotide sequence encoding a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity, wherein theamino acid sequence of the polypeptide has at least 95% sequenceidentity to the amino acid sequence set forth in SEQ ID NOs: 54, 61, 62,63, 64, 65, 66, 67, 68, 69 or 70; and (xvi) a nucleotide sequence thatis fully complementary to the nucleotide sequence of any one of(xi)-(xv); wherein the first recombinant nucleic acid molecule, thesecond recombinant nucleic acid molecule and the third recombinantnucleic acid molecule are stably incorporated into the genome of thetransformed plant.

In another embodiment, a transformed plant comprising in its genome atleast one recombinant nucleic acid molecule seclected from the groupconsisting of the first recombinant nucleic acid molecule, the secondrecombinant nucleic acid molecule and the third recombinant nucleic acidmolecule of above, wherein the at least one recombinant nucleic acidmolecule is stably incorporated into the genome of the transformedplant.

In another embodiment, the transformed plant may be a monocot selectedfrom the group consisting of maize, wheat, rice, sorghum, barley, milletand rye.

In another embodiment, the transformed plant may be a dicot selectedfrom the group consisting of soybean, Brassica sp., alfalfa, safflower,sunflower, cotton, peanut, canola, Arabidopsis, tobacco and potato.

In another embodiment, the at least one regulatory sequence of thefirst, second or third recombinant nucleic acid molecule comprises atleast one promoter selected from the group consisting of seed-preferred,constitutive, chemically regulated, tissue-preferred, anddevelopmentally regulated promoters.

In another embodiment, the invention includes seed of the transformedplant, wherein said seed comprises in its genome the first recombinantnucleic acid molecule, the second recombinant nucleic acid molecule andthe third recombinant nucleic acid molecule.

In another embodiment, the transformed plant of the invention produces aseed with an increased level of alpha-tocotrienol, beta-tocotrienol, orboth, relative to a plant with a similar genetic background but lackingsaid first recombinant nucleic acid molecule, said second recombinantnucleic acid molecule and said third recombinant nucleic acid molecule.

In another embodiment, the invention includes a method of increasing thelevel of alpha-tocotrienol, beta-tocotrienol, or both, in a plant,comprising stably incorporating into a plant genome: (a) a firstrecombinant nucleic acid molecule comprising at least one regulatorysequence operably linked to at least one nucleotide sequence selectedfrom the group consisting of: (i) a nucleotide sequence encoding apolypeptide having gamma-tocopherol methyltransferase activity; (ii) anucleotide sequence set forth in SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37 or 39; (iii) a nucleotide sequence encodingthe amino acid sequence set forth in SEQ ID NOs: 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38 or 40; (iv) a nucleotide sequence havingat least 80% sequence identity to the nucleotide sequence set forth inany one of (i)-(iii), wherein the nucleotide sequence encodes apolypeptide having gamma-tocopherol methyltransferase activity; and (v)a nucleotide sequence that is complementary to the nucleotide sequenceof any one of (i)-(iv); (b) a second recombinant nucleic acid moleculecomprising at least one regulatory sequence operably linked to at leastone nucleotide sequence selected from the group consisting of: (vi) anucleotide sequence encoding a polypeptide having homogentisategeranylgeranyl transferase activity; (vii) a nucleotide sequence setforth in SEQ ID NOs: 1, 3, 5, 7, or 9; (viii) a nucleotide sequenceencoding the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, or10; (ix) a nucleotide sequence having at least 80% sequence identity tothe nucleotide sequence set forth in any one of (vi)-(viii), wherein thenucleotide sequence encodes a polypeptide having homogentisategeranylgeranyl transferase activity; and (x) a nucleotide sequence thatis complementary to the nucleotide sequence of any one of (vi)-(ix); and(c) a third recombinant nucleic acid molecule comprising at least oneregulatory sequence operably linked to at least one nucleotide sequenceselected from the group consisting of: (xi) a nucleotide sequenceencoding a polypeptide having 2-methyl-6-phytylbenzoquinolmethyltransferase activity; (xii) a nucleotide sequence set forth in SEQID NO:53; (xiii) a nucleotide sequence encoding the amino acid sequenceset forth in SEQ ID NOs: 54, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70;(xiv) a nucleotide sequence having at least 80% sequence identity to thenucleotide sequence set forth in any one of (xi)-(xii), wherein thenucleotide sequence encodes a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity; (xv) anucleotide sequence encoding a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity, wherein theamino acid sequence of the polypeptide has at least 95% sequenceidentity to the amino acid sequence set forth in SEQ ID NOs: 54, 61, 62,63, 64, 65, 66, 67, 68, 69 or 70; and (xvi) a nucleotide sequence thatis fully complementary to the nucleotide sequence of any one of(xi)-(xv); and selecting a transformed plant that has an increased levelof alpha-tocotrienol, beta-tocotrienol, or both, relative to a plantwith a similar genetic background but lacking said first recombinantnucleic acid molecule, said second recombinant nucleic acid molecule andsaid third recombinant nucleic acid molecule.

In another embodiment, a method in which the first, second and thirdrecombinant nucleic acid molecules are incorporated into the plantgenome by co-transformation of a plant cell.

In another embodiment, a method in which at least one of said first,second or third recombinant nucleic acid molecules is incorporated intothe plant genome by re-transformation of a transformed plant cell,wherein said transformed plant cell comprises at least one of saidfirst, second or third recombinant nucleic acid molecules.

In another embodiment, a method in which at least one of said first,second or third recombinant nucleic acid molecule is incorporated intothe plant genome by breeding.

In another embodiment, a method in which the at least one regulatorysequence of the first, second or third recombinant nucleic acid moleculecomprises at least one promoter selected from the group consisting ofseed-preferred, constitutive, chemically regulated, tissue-preferred,and developmentally regulated promoters.

In another embodiment, the invention includes methods for transformingplants and plants cells to change the oil content therein comprisingtransforming a plant with one to three nucleotide sequences alone or inany combination of two or three nucleotide sequences. The methodcomprises; (a) obtaining a first plant comprising in its genome a firstrecombinant nucleic acid molecule comprising at least one regulatorysequence operably linked to a nucleotide sequence encoding a polypeptidehaving gamma-tocopherol methyltransferase activity; and (b) crossing thetransgenic plant of step (a) with a second plant comprising in itsgenome a second recombinant nucleic acid molecule comprising at leastone regulatory sequence operably linked to a nucleotide sequenceencoding a polypeptide having homogentisate geranylgeranyl transferaseactivity; (c) crossing the transgenic plant of step (b) with a thirdplant comprising in its genome a third recombinant nucleic acid moleculecomprising at least one regulatory sequence operably linked to anucleotide sequence encoding a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity; (d) obtaining aprogeny plant from said step (c) crossing, wherein said progeny plantcomprises in its genome the first recombinant nucleic acid molecule, thesecond recombinant nucleic acid molecule and the third recombinantnucleic acid molecule, and wherein said progeny plant exhibits anincreased level of alpha-tocotrienol, beta-tocotrienol, or both,relative to a plant with a similar genetic background but lacking saidfirst recombinant nucleic acid molecule, said second recombinant nucleicacid molecule and said third recombinant nucleic acid molecule.

In another embodiment, a method of increasing the level ofalpha-tocotrienol, beta-tocotrienol, or both, in a plant, comprising:(a) obtaining a first transformed plant comprising in its genome a firstrecombinant nucleic acid molecule comprising at least one regulatorysequence operably linked to at least one nucleotide sequence selectedfrom the group consisting of: (i) a nucleotide sequence encoding apolypeptide having gamma-tocopherol methyltransferase activity; (ii) anucleotide sequence set forth in SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37 or 39; (iii) a nucleotide sequence encodingthe amino acid sequence set forth in SEQ ID NOs: 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38 or 40; (iv) a nucleotide sequence havingat least 80% sequence identity to the entire coding sequence of thenucleotide sequence set forth in any one or (i)-(iii), wherein thenucleotide sequence encodes a polypeptide having gamma-tocopherolmethyltransferase activity; and (v) a nucleotide sequence that iscomplementary to the nucleotide sequence of any one of (i)-(iv); (b)crossing the transformed plant of step (a) with a second transformedplant comprising within its genome a second recombinant nucleic acidmolecule comprising at least one regulatory sequence operably linked toat least one nucleotide sequence selected from the group consisting of:(vi) a nucleotide sequence encoding a polypeptide having homogentisategeranylgeranyl transferase activity; (vii) a nucleotide sequence setforth in SEQ ID NOs: 1, 3, 5, 7 or 9; (viii) a nucleotide sequenceencoding the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8 or10; (ix) a nucleotide sequence having at least 80% sequence identity tothe entire coding sequence of the nucleotide sequence set forth in(vi)-(viii), wherein the nucleotide sequence encodes a polypeptidehaving homogentisate geranylgeranyl transferase activity; and (x) anucleotide sequence that is complementary to the nucleotide sequence ofany one of (vi)-(ix); and (c) crossing the transformed plant of step (b)with a third transformed plant comprising within its genome a thirdrecombinant nucleic acid molecule comprising at least one regulatorysequence operably linked to at least one nucleotide sequence selectedfrom the group consisting of: (xi) a nucleotide sequence encoding apolypeptide having 2-methyl-6-phytylbenzoquinol methyltransferaseactivity; (xii) a nucleotide sequence set forth in SEQ ID NO:53; (xiii)a nucleotide sequence encoding the amino acid sequence set forth in SEQID NOs: 54 or 70; (xiv) a nucleotide sequence having at least 80%sequence identity to the nucleotide sequence set forth in any one of(xi)-(xii), wherein the nucleotide sequence encodes a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity; (xv) anucleotide sequence encoding a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity, wherein theamino acid sequence of the polypeptide has at least 95% sequenceidentity to the amino acid sequence set forth in SEQ ID NOs: 54, 61, 62,63, 64, 65, 66, 67, 68, 69 or 70; and (xvi) a nucleotide sequence thatis fully complementary to the nucleotide sequence of any one of(xi)-(xv); and selecting a progeny plant from said step (c) crossing,wherein said progeny plant comprises in its genome the first recombinantnucleic acid molecule, the second recombinant nucleic acid molecule andthe third recombinant nucleic acid molecule, and wherein said progenyplant exhibits an increased level of alpha-tocotrienol,beta-tocotrienol, or both, relative to a plant with a similar geneticbackground but lacking said first recombinant nucleic acid molecule,said second recombinant nucleic acid molecule and said third recombinantnucleic acid molecule.

In another embodiment, any of the methods of the invention wherein theplant is a monocot is selected from the group consisting of maize,wheat, rice, sorghum, barley, millet and rye.

In another embodiment, any of the methods of the invention wherein theplant is a dicot is selected from the group consisting of soybean,Brassica sp., alfalfa, safflower, sunflower, cotton, peanut, canola,Arabidopsis, tobacco and potato.

In another embodiment, the invention includes transformed seed orbyproducts of any of the transformed plants of the invention.

In another embodiment, the transformed seed of the invention has analpha-tocotrienol level of at least 20 parts per million (ppm).

In another embodiment, the transformed seed of the invention containsalpha-tocotrienol in an amount of at least 20% of total tocopherol andtocotrienol content in the transformed seed.

In another embodiment, the transformed seed of the invention has analpha-tocotrienol content of at least 70% of total combined tocopheroland tocotrienol content in the transformed seed.

In another embodiment, the transformed seed of the invention contains acombined level of alpha-tocotrienol and alpha-tocopherol of at least 95%of total tocopherol and tocotrienol content in the transformed seed.

In another embodiment, a method of improving the tissue quality of ananimal, comprising feeding the animal the transformed seed of theinvention.

In another embodiment, the tissue is meat and the quality of the meat ismeasured by at least one criteria selected from the group consisting ofincreased pH, improved food color, improved oxidative stability,increased shelf life and reduced purge.

In another embodiment, the animal is a ruminant, preferably cattle.

In another embodiment, the animal is a non-ruminant, preferably swine orpoultry.

In another embodiment, an isolated polynucleotide comprising SEQ IDNO:57.

In another embodiment, an isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity, wherein thepolypeptide has an amino acid sequence of at least 95% sequenceidentity, based on the Clustal W method of alignment, when compared toSEQ ID NO:54 or 70, or (b) a complement of the nucleotide sequence,wherein the complement and the nucleotide sequence consist of the samenumber of nucleotides and are 100% complementary. The amino acidsequence of the polypeptide preferably comprises SEQ ID NO:54 or 70. Thenucleotide sequence preferably comprises SEQ ID NO:53.

In another embodiment, the present invention includes a vectorcomprising any of the isolated polynucleotides of the present invention.

In another embodiment, the present invention includes a recombinant DNAconstruct comprising any of the isolated polynucleotides of the presentinvention operably linked to at least one regulatory sequence.

In another embodiment, the present invention concerns a method fortransforming a cell comprising transforming a cell with any of theisolated polynucleotides of the present invention. The cell transformedby this method is also included. In particular embodiments, the cell iseukaryotic cell, e.g., a yeast, insect or plant cell, or prokaryotic,e.g., a bacterium.

In another embodiment, the present invention includes a method forproducing a transgenic plant comprising transforming a plant cell withany of the isolated polynucleotides or recombinant DNA constructs of thepresent invention and regenerating a transgenic plant from thetransformed plant cell. The invention is also directed to the transgenicplant produced by this method, and transgenic seed obtained from thistransgenic plant.

In another embodiment, an isolated polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity, wherein thepolypeptide has an amino acid sequence of at least 95% sequenceidentity, based on the Clustal W method of alignment, when compared toone of SEQ ID NO:54 or 70. The amino acid sequence of the polypeptideprefereably comprises SEQ ID NO:54 or 70.

In another embodiment, a method for isolating a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity comprisingisolating the polypeptide from a cell or culture medium of the cell,wherein the cell comprises a recombinant DNA construct comprising thepolynucleotide of the invention operably linked to at least oneregulatory sequence.

In another embodiment, a method of altering the level of expression of a2-methyl-6-phytylbenzoquinol methyltransferase in a host cellcomprising: (a) transforming a host cell with the recombinant DNAconstruct of the invention; and (b) growing the transformed host cellunder conditions that are suitable for expression of the recombinant DNAconstruct wherein expression of the recombinant DNA construct results inproduction of altered levels of the 2-methyl-6-phytylbenzoquinolmethyltransferase in the transformed host cell.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIG. 1 is a schematic depiction of the tocopherol biosynthetic pathway.

FIG. 2 is a schematic depiction of the tocotrienol biosynthetic pathway.

FIG. 3A-3C show a multiple alignment of the 2-methyl-6-phytylbenzoquinolmethyltransferase polypeptides of SEQ ID NOs: 54, 61, 62, 63, 64, 65,66, 67, 68, 69 and 70. The multiple alignment was assembled using theClustal W method of alignment with the default parameters. Residues thatmatch SEQ ID NO:54 exactly are enclosed in a box. Above the alignment isshown a consensus sequence. A residue is shown in the consensus sequencewhen all residues at that position are identical.

FIG. 4 shows the percent sequence identity and divergence for each pairof polypeptides from the multiple alignment of FIG. 3A-3C.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Many modifications and other embodiments of the invention set forthherein will come to mind to one skilled in the art to which thisinvention pertains, having the benefit of the teachings presented in thedescriptions and the drawings herein. Therefore, it is to be understoodthat the invention is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims. Although specificterms are employed herein, they are used in a generic and descriptivesense only and not for purposes of limitation.

The articles “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more than one element.

The combination of HGGT, gamma-tocopherol methyltransferase and2-methyl-6-phytylbenzoquinol methyltransferase polynucleotides may beused in plants, plant cells, yeast, and microbes to alter the tocols,such as tocotrienols, produced in the cells via the production of therespective enzymes from each polynucleotide. The instant inventionshows, inter alia, that the combination of HGGT, gamma-tocopherolmethyltransferase and 2-methyl-6-phytylbenzoquinol methyltransferasepolynucleotides, more specifically producing the enzymes they encode,may be used to significantly increase the content of vitamin E-relatedantioxidants, specifically alpha- and beta-tocotrienol, in edibletissues of vegetable, fruit, and agronomic crop plants, including grainswhich include but are not limted to maize and soybean seed. The changesin vitamin-E antioxidant content will also be reflected in the oilobtained from these plants, grains and seeds. The use of polynucleotidesencoding HGGT and gamma-tocopherol methyltransferase is described inU.S. Patent Application Publication No. 2007-0199096, which is hereinincorporated by reference.

The invention includes compositions and methods for altering tocols. Thecompositions and methods find use in improving the antioxidant qualityof grain for use as food for humans and feed for livestock. Furthermore,the tocols can be extracted, purified or further altered via processing.

As used herein, “grain” means the mature seed produced by commercialgrowers for purposes other than reproducing the species and/or immatureseed as an integral part of whole plant maize harvested for silage. Asused herein, grain includes plant parts commonly categorized as a fruit,nut or vegetable.

As used herein, “wild-type” refers to untransformed organisms anddescendants of untransformed organisms.

The molecular formula of a chemical may be presented in various formats.For example, the terms “ZnSO₄.7H₂O”, “ZnSO₄.7H₂O”, “ZnSO₄*7H₂O”, and“ZnSO₄-7H₂O” are used interchangeably herein.

The term “tocol” refers generally to any of the tocopherol andtocotrienol molecular species (e.g., α-, β-, γ-, and δ-) that are knownto occur in biological systems. The term “tocol content” refers to thetotal amount of tocopherol and tocotrienol in a whole plant, tissue, orcell or in a microbial host. The term “tocol composition” refers both tothe ratio of the various tocols produced in any given biological systemand to characteristics, such as antioxidant activity, of any one tocolcompound. When the alteration of tocols is taught or claimed herein,such alteration can be to tocol content and/or tocol composition. Whenan increase of tocols is taught or claimed herein, such increase refersto an increase of tocol content and/or an increase of tocol activity.

The term “tocotrienol” refers generally to any of the tocotrienolmolecular species (e.g., α, β, γ, and δ) that are known to occur inbiological systems. The term “tocotrienol content” refers to the totalamount of tocotrienol in a whole plant, tissue, or cell or in amicrobial host. The term “tocotrienol composition” refers both to theratio of the various tocotrienols produced in any given biologicalsystem and to characteristics, such as antioxidant activity, of any onetocotrienol compound. When the alteration of a tocotrienol is taught orclaimed herein, such alteration can be to tocotrienol content and/ortocotrienol composition. When an increase of tocotrienols is taught orclaimed herein, such increase refers to an increase of tocotrienolcontent and/or an increase of tocotrienol activity.

The term “homogentisate phytyltransferase” or “HPT” refers to the enzymethat catalyzes the condensation of homogentisate (or homogentisic acid)and phytyl pyrophosphate (or phytyl diphosphate). This reaction isbelieved to be the committed step in tocopherol biosynthesis. Othernames that have been used to refer to this enzyme include “homogentisatephytyl pyrophosphate prenyltransferase” and “homogentisate phytyldiphosphate prenyltransferase”. The shortened version phytyl/prenyltransferase is also used.

The terms “homogentisate geranylgeranyl transferase” and “HGGT”, whichare used interchangeably herein, refer to the enzyme that catalyzes thecondensation of homogentisate (or homogentisic acid) and geranylgeranylpyrophosphate (or geranylgeranyl diphosphate). This reaction is animportant step in tocotrienol biosynthesis and can result in thealteration of the tocol content and/or composition. HGGT enzymes mayinclude, but are not limited to, those shown in Table 1.

TABLE 1 Homogentisate Geranylgeranyl Transferase Enzymes SEQ ID NO:(Nucle- (Amino Protein Clone Designation otide) Acid) barleyhomogentisate bdl2c.pk006.o2 1 2 geranylgeranyl transferase wheathomogentisate wdk2c.pk012.f2:cgs 3 4 geranylgeranyl transferase ricehomogentisate rds1c.pk007.m9 5 6 geranylgeranyl transferase maizehomogentisate cco1n.pk087.l17:cgs 7 8 geranylgeranyl transferase maizehomogentisate p0058.chpbj67r:fis 9 10 geranylgeranyl transferase

The terms “gamma-tocopherol methyltransferase”, “γ-TMT”, “GTMT” and“VTE4”, which are used herein, refer to the enzyme that catalyzes themethylation of gamma- and delta-tocopherol to alpha- andbeta-tocopherol, respectively, and to the methylation of gamma- anddelta-tocotrienol to alpha- and beta-tocotrienol, respectively. Thisreaction is an important step in tocotrienol biosynthesis and can resultin the alteration of the tocol content and/or composition.Gamma-tocopherol methyltransferase enzymes may include, but are notlimited to, those shown in Table 2.

TABLE 2 Gamma-Tocopherol Methyltransferase Enzymes Clone Designation SEQID NO: or GenBank Accession (Nucle- (Amino Protein No. otide) Acid)Soybean gamma-tocopherol sah1c.pk004.g2 11 12 Methyltransferase Soybeangamma-tocopherol sah1c.pk001.k8:fis 13 14 Methyltransferase maizegamma-tocopherol p0060.coran49r:fis 15 16 methyltransferase wheatgamma-tocopherol wr1.pk0077.fl:fis 17 18 methyltransferase lotuscorniculatus gamma- GenBank Accession 19 20 tocopherol methyltransferaseNo. DQ13360 soybean gamma-tocopherol GenBank Accession 21 22methyltransferase No. AY960126 rice gamma-tocopherol GenBank Accession23 24 methyltransferase No. XM467331 Brassica gamma-tocopherol GenBankAccession 25 26 Methyltransferase No. AF381248 Perilla frutescens gamma-GenBank Accession 27 28 tocopherol methyltransferase No. AF213481Arabidopsis thaliana GenBank Accession 29 30 gamma-tocopherol No.AF104220 methyltransferase Medicago truncatula gamma- GenBank Accession31 32 tocopherol Methyltransferase No. AY962639 Chlamydomonas gamma-GenBank Accession 33 34 tocopherol methyltransferase No. AJ884948Synechocystis gamma- GenBank Accession 35 36 tocopherolmethyltransferase No. NP_442492 Anabaena gamma- GenBank Accession 37 38tocopherol Methyltransferase No. BAB73502 Gloeobacter violaceus GenBankAccession 39 40 gamma-tocopherol No. NP_926036 methyltransferase

Limited information regarding enzymes catalyzing methylations of gamma-and delta-tocotrienol is available. U.S. Application No. 2003154513discloses sequences derived from cotton, maize and the cyanobacteriaAnabaena. These sequences show similarity to gamma-tocopherolmethyltransferase genes from Arabidopsis (PCT Publication No. WO99/04622) and soybean (PCT Publication No. WO 00/032757). Theheterologously expressed enzyme from maize, a moncotyledoneous plant,showed almost equal activity with tocopherol and tocotrienol substrates.On the other hand, gamma-tocopherol methyltransferase orthologs from thedicotyledoneous plant cotton or blue-green algae showed only traceactivities with tocotrienol substrates.

The terms “2-methyl-6-phytylbenzoquinol methyltransferase”, “VTE3” and“MPBQMT”, which are used interchangeably herein, refer to the enzymethat catalyzes the methylation of 2-methyl-6-phytylbenzoquinol (MPBQ)prior to cyclization. This reaction is an important step in tocotrienolbiosynthesis and can result in the alteration of the tocol contentand/or composition. 2-methyl-6-phytylbenzoquinol methyltransferaseenzymes may include, but are not limited to, those shown in Table 3. Theamino acid sequence of the enzyme in which the putative transit peptidehas been removed is designated as “mature”.

TABLE 3 2-Methyl-6-Phytylbenzoquinol Methyltransferase Enzymes CloneName, NCBI GI No. SEQ ID or Patent Reference Plant NO fds1n.pk003.e5(FIS) Momordica charantia 54 GI No. 108385436 Arabidopsis 61 GI No.157348021 Grape 62 GI No. 80971672 Sunflower 63 US2007061916 Cotton 64WO2003034812 Soybean 65 WO2003034812 Corn 66 GI No. 108385436-derivedArabidopsis (mature) 67 WO2003034812 Soybean (mature) 68 WO2003034812Corn (mature) 69

The VTE3 (vitamin E defective) locus in Arabidopsis has been isolatedand characterized, and encodes the Arabidopsis2-methyl-6-phytylbenzoquinol methyltransferase (Cheng et al. 2003 PlantCell 15:2343-2356). Recombinant DNA constructs encoding the ArabidopsisVTE3 and VTE4 polypeptides have been co-expressed in transgenic soybean(Van Eenennaam et al. 2003 Plant Cell 15:3007-3019).

The present invention concerns an isolated polynucleotide comprising anucleotide sequence encoding a 2-methyl-6-phytylbenzoquinolmethyltransferase polypeptide having at least 95% identity, based on theClustal W method of alignment, when compared to SEQ ID NO:54 or 70.

The invention includes the use of the combination of HGGT,gamma-tocopherol methyltransferase and 2-methyl-6-phytylbenzoquinolmethyltransferase enzymes to significantly increase the content ofvitamin E-related antioxidants, specifically alpha- andbeta-tocotrienol, in organisms including plants and microorganisms. Theinvention is not limited to the disclosed embodiments, but encompassesall enzymes which include these activities.

This invention also includes to the isolated complement of suchpolynucleotides, wherein the complement and the polynucleotide consistof the same number of nucleotides, and the nucleotide sequences of thecomplement and the polynucleotide have 100% complementarity.

In another embodiment, this invention concerns viruses and host cellscomprising either the recombinant DNA constructs of the invention asdescribed herein or isolated polynucleotides of the invention asdescribed herein. Examples of host cells which can be used to practicethe invention include, but are not limited to, yeast, bacteria, andplants.

In another embodiment, the present invention concerns a2-methyl-6-phytylbenzoquinol methyltransferase polypeptide having anamino acid sequence that is at least 95% identical, based on the ClustalW method of alignment, to a polypeptide of SEQ ID NO:54 or 70.

As was noted above, the nucleic acid fragments of the instant inventionmay be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the level of tocol content and/orcomposition in those cells.

The invention provides isolated nucleotide molecules comprising thecombination of nucleotide sequences encoding HGGT, gamma-tocopherolmethyltransferase and 2-methyl-6-phytylbenzoquinol methyltransferase.Also provided are isolated polypeptides encoded by such nucleotidesequences. The nucleotide sequences find use in methods for alteringalpha- and beta-tocotrienols in a biological system such as a plant. Themethods include improving the antioxidant activity of grain, alteringtocotrienols in a plant or part thereof, and improving tocols in a host.The methods comprise transforming a plant or host with at least onenucleotide construct comprising at least a portion of at least onenucleotide sequence encoding HGGT, at least a portion of at least onenucleotide sequence encoding gamma-tocopherol methyltransferase and atleast a portion of at least one nucleotide sequence encoding2-methyl-6-phytylbenzoquinol methyltransferase. If desired, thenucleotide construct may additionally comprise at least one operablylinked regulatory sequence that drives expression in the plant ofinterest. Such a nucleotide construct can be used to increase theexpression of HGGT, gamma-tocopherol methyltransferase and2-methyl-6-phytylbenzoquinol methyltransferase.

Also provided are novel compositions of seed and extracted oils. Seedand extracted oils are provided that have unexpectantly high levels ofalpha-tocotrienol, beta-tocotrienol, or both. Seed or oil with highlevels of alpha-tocotrienol have better bioavailabilty ofalpha-tocotrienol as compared to other tocotrienol species (Kiyose etal. (2004) J. Clin. Biochem. Nutr. 35(1):47-52, entitiled—Distributionand metabolism of tocopherols and tocotrienols in vivo).

Methods of isolating seed oils are well known in the art: (Young et al.,Processing of Fats and Oils, In The Lipid Handbook, Gunstone et al.,eds., Chapter 5 pp 253-257; Chapman & Hall: London (1994)). For example,soybean oil is produced using a series of steps involving the extractionand purification of an edible oil product from the oil-bearing seed.Soybean oils and soybean byproducts are produced using the generalizedsteps shown in Table 4.

TABLE 4 Generalized Steps for Soybean Oil and By-product ProductionProcess Impurities Removed and/or Step Process By-Products Obtained # 1soybean seed # 2 oil extraction Meal # 3 degumming Lecithin # 4 alkalior physical refining gums, free fatty acids, pigments # 5 water washingSoap # 6 bleaching color, soap, metal # 7 (hydrogenation) # 8(winterization) Stearine # 9 deodorization free fatty acids,tocopherols, sterols, volatiles # 10  oil products

More specifically, soybean seeds are cleaned, tempered, dehulled andflaked, thereby increasing the efficiency of oil extraction. Oilextraction is usually accomplished by solvent (e.g., hexane) extractionbut can also be achieved by a combination of physical pressure and/orsolvent extraction. The resulting oil is called crude oil. The crude oilmay be degummed by hydrating phospholipids and other polar and neutrallipid complexes that facilitate their separation from the nonhydrating,triglyceride fraction (soybean oil). The resulting lecithin gums may befurther processed to make commercially important lecithin products usedin a variety of food and industrial products as emulsification andrelease (i.e., antisticking) agents. Degummed oil may be further refinedfor the removal of impurities (primarily free fatty acids, pigments andresidual gums). Refining is accomplished by the addition of a causticagent that reacts with free fatty acid to form soap and hydratesphosphatides and proteins in the crude oil. Water is used to wash outtraces of soap formed during refining. The soapstock byproduct may beused directly in animal feeds or acidulated to recover the free fattyacids. Color is removed through adsorption with a bleaching earth thatremoves most of the chlorophyll and carotenoid compounds. The refinedoil can be hydrogenated, thereby resulting in fats with various meltingproperties and textures. Winterization (fractionation) may be used toremove stearine from the hydrogenated oil through crystallization undercarefully controlled cooling conditions. Deodorization (principally viasteam distillation under vacuum) is the last step and is designed toremove compounds which impart odor or flavor to the oil. Other valuablebyproducts such as tocopherols and sterols may be removed during thedeodorization process. Deodorized distillate containing these byproductsmay be sold for production of natural vitamin E and other high-valuepharmaceutical products. Refined, bleached, (hydrogenated, fractionated)and deodorized oils and fats may be packaged and sold directly orfurther processed into more specialized products. A more detailedreference to soybean seed processing, soybean oil production andbyproduct utilization can be found in Erickson, Practical Handbook ofSoybean Processing and Utilization, The American Oil Chemists' Societyand United Soybean Board (1995).

Among the many applications of improved tocols, tocotrienols andantioxidant activity are improved storage of grain, improved stabilityof oil extracted from grain, benefits to humans consuming the grain,improved meat quality from animals consuming the grain, and theproduction of novel tocols or tocotrienols for cosmetic, industrialand/or nutraceutical use (U.S. Application No. 2004266862; Karunanandaaet al. (2005) Metab. Eng. 7:384-400). It is also known that the presenceof tocols in plant vegetative green tissue such as leaf tissue isnecessary to protect the plant from the photo-oxidative damage induceddirectly and indirectly by the production of free oxygen radicals in thechloroplast during oxygenic photosynthesis. It is therefore likely thatectopic expression of tocotrienols in green plant tissue, such as leaftissue, in addition to the normal tocopherol content of the leaf willlead to an increase ability to withstand such photo-oxidative damage,and thus lead to an increase in the photosynthetic capacity of theplant. This would translate to an increase in harvestable yield for theplant over the entire growing season.

The nucleotide construct of the invention may additionally comprise atleast one regulatory sequence that drives expression in a host or plant.Optional regulatory sequences include, for maize, an embryo preferredpromoter such as promoters for the 16 kDa and 18 kDa oleosin genes, anendosperm preferred promoter, such as the promoter for the 10 kDa zeingene, and a vegetative promoter such as promoters for ubiquitin genes.

If desired, two or more of such nucleotide sequences may be linked orjoined together to form one polynucleotide molecule, and such apolynucleotide may be used to transform a plant. For example, anucleotide construct comprising a nucleotide sequence encoding an HGGTcan be linked with another nucleotide sequence encoding the same oranother HGGT. Nucleotide sequences encoding both HGGT andgamma-tocopherol methyltransferase may also be linked in a nucleotideconstruct. Additionally, nucleotide sequences encoding HGGT,gamma-tocopherol methyltransferase and 2-methyl-6-phytylbenzoquinolmethyltransferase may also be linked in a nucleotide construct.Similarly, the three nucleotide sequences can be provided on differentnucleotide constructs, and each of the separate nucleotide sequences canbe operably linked to at least one regulatory sequence that drivesexpression in a plant. For example, a construct may be used thatincreases total HGGT activity and decreases total HPT activity, therebyresulting in shunting the pathway towards the production of tocotrienolsand decreased production of tocopherols.

An alternative strategy may also be used. If separate nucleotideconstructs are employed for an HGGT nucleotide sequence, agamma-tocopherol methyltransferase nucleotide sequence and a2-methyl-6-phytylbenzoquinol methyltransferase nucleotide sequence,three individual plants may be transformed with the nucleotideconstructs, and the plants may then be crossed to produce progeny havingthe desired genotype of HGGT, gamma-tocopherol methyltransferase and2-methyl-6-phytylbenzoquinol methyltransferase nucleotide sequences(i.e., also referred to as genetic stacks).

Additionally, a construct to down-regulate the geranylgeranyl reductaseresponsible for producing phytol pyrophosphosphate, one of theprecursors for tocopherol biosynthesis, may be linked in cis with aconstruct to express HGGT. The result of this manipulation would be anincreased pool size of geranylgeranyl-pyrophosphate and a correspondingincrease of flux into the tocotrienol biosynthetic pathway. Flux intotocotrienols can also be increased by increasing flux of carbon into theshikimate pathway and non-mevalonate pathway of isoprenoid biosynthesis.Specifically, this flux can be accomplished through chloroplast-targetedexpression of genes such as bifunctional chorismate mutase-prephenatedehydrogenase (TYRA) (from bacteria) and p-hydroxyphenylpyruvatedioxygenase (HPPD) genes from plants (Karunanandaa et al. (2005) Metab.Eng. 7:384-400).

Nucleic acid molecules of the present invention are preferablyrecombinant nucleic acid molecules (or may also be referred to asrecombinant DNA constructs). As used herein, “recombinant” refers to anartificial combination of two otherwise separated segments of sequence,e.g., by chemical synthesis or by the manipulation of isolated segmentsof nucleic acids by genetic engineering techniques. “Recombinant” alsoincludes reference to a cell or vector, that has been modified by theintroduction of a heterologous nucleic acid or a cell derived from acell so modified, but does not encompass the alteration of the cell orvector by naturally occurring events (e.g., spontaneous mutation,natural transformation/transduction/transposition) such as thoseoccurring without deliberate human intervention.

As used herein, “recombinant DNA construct” refers to a combination ofnucleic acid fragments that are not normally found together in nature.Accordingly, a recombinant DNA construct may comprise regulatorysequences and coding sequences that are derived from different sources,or regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different than that normally found innature. The terms “recombinant DNA construct” and “recombinant nucleicacid molecule” are used interchangeably herein.

The methods of the present invention can be employed to alter tocols ortocotrienols in any plant or part thereof, and antioxidant activity maythereby be altered. Plants that may be used in the invention include,but are not limited to, field crops (e.g., alfalfa, barley, bean, maize,canola, cotton, flax, pea, rice, rye, safflower, sorghum, oats, millet,soybean, sunflower, tobacco, and wheat); vegetable crops (e.g.,asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery,cucumber, eggplant, lettuce, onion, pepper, potato, pumpkin, radish,spinach, squash, taro, tomato, and zucchini); and fruit and nut crops(e.g., almond, apple, apricot, banana, blackberry, blueberry, cacao,cherry, coconut, cranberry, date, fajoa, filbert, grape, grapefruit,guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya,passion fruit, peach, peanut, pear, pineapple, pistachio, plum,raspberry, strawberry, tangerine, walnut, and watermelon) andArabidopsis. Some methods of the invention involve altering theantioxidant levels in grain and other parts of a plant that may besubjected to post-harvest processing. With post-harvest processing, thetocols or tocotrienols so produced can be a valuable source of recoveryfor millers and other processors.

Grain or vegetable oil derived from transgenic plants containingelevated levels of alpha- and beta-tocotrienol may be fed to livestockand poultry to improve the oxidative stability of meat products.Examples of improvements with practical benefit include increased colorstability of fresh beef during retail display and enhanced flavorstability of precooked meat products stored under refrigeration. Theseand other quality-related improvements may be expected becausetocotrienols function as chain-breaking free radical scavengers inmuscle tissue, and thus reduce oxidative reactions that degrade meatquality and reduce shelf life.

For example, improved beef quality can be demonstrated by feeding cattlea diet formulated with at least about 300-ppm of total alpha- andbeta-tocotrienol obtained from high-tocotrienol transgenic grain orvegetable oil for at least 100 days. For comparison, a group of cattlereared on a standard diet (no additional tocotrienol) under otherwiseidentical conditions can serve as the control treatment (“controlgroup”). To assess fresh meat color stability, ribeye steaks harvestedfrom each animal are individually packaged in foam trays with PVCoverwrap and placed under simulated retail display for seven days. Freshsteak color is subjectively evaluated by trained panelists on a gradedscale for visual color intensity and discoloration. Color is alsoevaluated instrumentally using a HunterLab MiniScan™ Spectrophotometeror similar device to assess the “a* value”, which is a measure of thedegree of redness. Results of these assays demonstrate that over timesteaks from cattle fed a high tocotrienol diet, on average, exhibitbetter subjective visual scores and higher (i.e., better) a*instrumental values than ribeye steaks from the control group over time.The improvement in color stability extends retail display time and thusreduces the amount of fresh product discounted and discarded due tocolor deterioration. Other fresh beef products, including ground beef,will also exhibit improved color stability with and thus provide asimilar benefit to retailers. (See also WO Publication No. 2005/002358,herein incorporated in its entirety by reference).

Methods for assessing tocopherol content and tocopherol composition(including tocopherol activity) are known in the art. Tocopherol contentand composition may be measured by HPLC in combination with fluorescencedetection. Such methods are described in numerous literature references(e.g., Kamal-Eldi A., Gorgen S., Pettersson J., Lampi A. M. (2000) J.Chromatogr. A 881:217-227; Bonvehi J. S., Coll F. V., Rius I. A. (2000)J. AOAC Intl. 83:627-634; Goffman F. D. and Böme T. (2001) J. Agric.Food Chem. 49:4990-4994). Such methods typically involve the resolutionof tocopherol molecular species contained in complex mixtures by use ofa normal or reverse phase HPLC matrix. Eluted tocopherol molecularspecies are then detected by fluorescence of the chromanol head groupwith an excitation wavelength typically in the range of 290 to 295 nmand an emission wavelength typically in the range of 325 to 335 nm.Using this methodology, the composition of a tocopherol mixture can bedetermined by comparing the retention times of separated molecularspecies with those of known standards. The content of each tocopherolmolecular species can be measured by the relative intensity of itsfluorescence emission at the selected wavelength. The absolute amount ofeach tocopherol species can be determined by measuring the intensity offluorescence emission relative to that of an internal standard, which isadded in a known amount to the tocopherol mixture prior to HPLCanalysis. A suitable internal standard can include a tocopherol analogthat is not normally found in nature (e.g., 5,7-dimethyltocol) or anaturally occurring tocopherol molecular species that is not present ina given tocopherol mixture. The total tocopherol content of a complexmixture of compounds can be derived by summing the absolute amount ofeach of the component tocopherol molecular species as determined by HPLCanalysis.

Methods for assessing tocotrienol content and tocotrienol composition(including tocotrienol activity) are known in the art. Tocotrienolcontent and composition may be measured by HPLC using methods describedabove for the analysis of tocopherol content and composition. Using HPLCtechniques described in Example 3 and elsewhere (e.g., Podda M., WeberC., Traber M. G., Packer L. (1996) J. Lipid Res. 37:893-901),tocotrienol molecular species can be readily resolved from tocopherolmolecular species in a complex mixture. The occurrence and structuralidentification of tocotrienols in a complex mixture can be determined bygas chromatography-mass spectrometry as described by Frega N., MozzonM., and Bocci F. (1998) J. Amer. Oil Chem. Soc. 75:1723-1728.

In addition, lipophilic antioxidant activity may be measured by assaysincluding the inhibition of the coupled auto-oxidation of linoleic acidand β-carotene and oxygen radical absorbance capacity (ORAC) asdescribed elsewhere (Serbinova E. A. and Packer L. (1994) Meth. Enzymol.234:354-366; Emmons C. L., Peterson D. M., Paul G. L. (1999) J. Agric.Food Chem. 47:4894-4898); Huang D et al (2002) J. Agric. Food Chem.).Such methods typically involve measuring the ability of antioxidantcompounds (i.e., tocols) in test materials to inhibit the decline offluorescence of a model substrate (fluorescein, phycoerythrin) inducedby a peroxyl radical generator(2′,2′-azobis[20amidinopropane]dihydrochloride).

The invention encompasses isolated or substantially purified nucleicacid or polypeptide compositions. An “isolated” or “purified” nucleicacid molecule or polypeptide, or biologically active portion thereof, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Preferably,an “isolated” nucleic acid is free of sequences (preferably proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid molecule cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, 0.3 kb or0.1 kb of nucleotide sequences that naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived. A polypeptide that is substantially free of cellular materialincludes preparations of polypeptide having less than about 30%, 20%,10%, 5%, (by dry weight) of contaminating polypeptide. When thepolypeptide of the invention or biologically active portion thereof isrecombinantly produced, preferably culture medium represents less thanabout 30%, 20%, 10%, 5%, 3% or 1% (by dry weight) of chemical precursorsor non-polypeptide-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences andpolypeptides encoded thereby are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence or a portion of the amino acid sequence. Functional fragmentsof a nucleotide sequence may encode polypeptide fragments that retainthe biological activity of the native protein and hence HGGT activityand/or gamma-tocopherol methyltransferase activity and/or2-methyl-6-phytylbenzoquinol methyltransferase activity. Alternatively,fragments of a nucleotide sequence that are useful as hybridizationprobes generally do not encode polypeptides retaining biologicalactivity. Thus, fragments of a nucleotide sequence may range from atleast about 20 nucleotides, about 30 nucleotides, about 50 nucleotides,about 70 nucleotides, about 100 nucleotides, about 150 nucleotides andup to the full-length nucleotide sequence encoding the polypeptides ofthe invention.

A fragment of a HGGT nucleotide sequence that encodes a biologicallyactive portion of an HGGT polypeptide of the invention will encode atleast 15, 25, 30, 50, 75, 100, or 125 contiguous amino acids, or up tothe total number of amino acids present in a full-length HGGTpolypeptide of the invention (for example, 407, 408, 404, 380 and 361amino acids for SEQ ID NO:2, 4, 6, 8 and 10, respectively). Fragments ofa HGGT nucleotide sequence that are useful as hybridization probes orPCR primers generally need not encode a biologically active portion ofan HGGT polypeptide.

Thus, a fragment of an HGGT nucleotide sequence may encode abiologically active portion of an HGGT polypeptide, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of an HGGTpolypeptide can be prepared by isolating a portion of one of the HGGTnucleotide sequences of the invention, expressing the encoded portion ofthe HGGT polypeptide (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the HGGT polypeptide.

Nucleic acid molecules that are fragments of an HGGT nucleotide sequencecomprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, or 700 nucleotides, or up to the number ofnucleotides present in a full-length HGGT nucleotide sequence disclosedherein (for example, 1457, 1365, 1242, 1730, and 1769 nucleotides forSEQ ID NO:1, 3, 5, 7 and 9, respectively).

Likewise, a fragment of a gamma-tocopherol methyltransferase nucleotidesequence that encodes a biologically active portion of agamma-tocopherol methyltranferase polypeptide of the invention willencode at least 15, 25, 30, 50, 75, 100, or 125 contiguous amino acids,or up to the total number of amino acids present in a full-lengthgamma-tocopherol methyltranferase polypeptide of the invention.Fragments of a gamma-tocopherol methyltranferase nucleotide sequencethat are useful as hybridization probes or PCR primers generally neednot encode a biologically active portion of a gamma-tocopherolmethyltransferase polypeptide.

Thus, a fragment of an gamma-tocopherol methyltranferase nucleotidesequence may encode a biologically active portion of an gamma-tocopherolmethyltranferase polypeptide, or it may be a fragment that can be usedas a hybridization probe or PCR primer using methods disclosed below. Abiologically active portion of an gamma-tocopherol methyltranferasepolypeptide can be prepared by isolating a portion of one of thegamma-tocopherol methyltranferase nucleotide sequences of the invention,expressing the encoded portion of the gamma-tocopherol methyltranferasepolypeptide (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of the gamma-tocopherol methyltranferasepolypeptide.

Nucleic acid molecules that are fragments of an gamma-tocopherolmethyltranferase nucleotide sequence comprise at least 16, 20, 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700nucleotides, or up to the number of nucleotides present in a full-lengthgamma-tocopherol methyltranferase nucleotide sequence disclosed herein.

Likewise, a fragment of a 2-methyl-6-phytylbenzoquinol methyltransferasenucleotide sequence that encodes a biologically active portion of a2-methyl-6-phytylbenzoquinol methyltransferase polypeptide of theinvention will encode at least 15, 25, 30, 50, 75, 100, or 125contiguous amino acids, or up to the total number of amino acids presentin a full-length 2-methyl-6-phytylbenzoquinol methyltransferasepolypeptide of the invention. Fragments of a2-methyl-6-phytylbenzoquinol methyltransferase nucleotide sequence thatare useful as hybridization probes or PCR primers generally need notencode a biologically active portion of a gamma-tocopherolmethyltransferase polypeptide.

Thus, a fragment of a 2-methyl-6-phytylbenzoquinol methyltransferasenucleotide sequence may encode a biologically active portion of a2-methyl-6-phytylbenzoquinol methyltransferase polypeptide, or it may bea fragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of a2-methyl-6-phytylbenzoquinol methyltransferase polypeptide can beprepared by isolating a portion of one of the2-methyl-6-phytylbenzoquinol methyltransferase nucleotide sequences ofthe invention, expressing the encoded portion of the2-methyl-6-phytylbenzoquinol methyltransferase polypeptide (e.g., byrecombinant expression in vitro) and assessing the activity of theencoded portion of the 2-methyl-6-phytylbenzoquinol methyltransferasepolypeptide.

Nucleic acid molecules that are fragments of an2-methyl-6-phytylbenzoquinol methyltransferase nucleotide sequencecomprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, or 700 nucleotides, or up to the number ofnucleotides present in a full-length 2-methyl-6-phytylbenzoquinolmethyltransferase nucleotide sequence disclosed herein.

By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the HGGT and/or gamma-tocopherolmethyltransferase and/or 2-methyl-6-phytylbenzoquinol methyltransferasepolypeptides of the invention. Naturally occurring allelic variants suchas these can be identified with the use of well-known molecular biologytechniques, as, for example, with polymerase chain reaction (PCR) andhybridization techniques as outlined below. Variant nucleotide sequencesalso include synthetically derived nucleotide sequences, such as thosegenerated, for example, by using site-directed mutagenesis but whichstill encode an HGGT and/or gamma-tocopherol methyltransferase and/or2-methyl-6-phytylbenzoquinol methyltransferase polypeptide of theinvention. Generally, variants of a particular nucleotide sequence ofthe invention will have at least about 80% generally at least about 85%,preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, andmore preferably at least about 98%, 99% or more sequence identity tothat particular nucleotide sequence as determined by sequence alignmentprograms described elsewhere herein using default parameters.

By “variant” polypeptide is intended a polypeptide derived from thenative polypeptide by deletion (so-called truncation) or addition of oneor more amino acids to the N-terminal and/or C-terminal end of thenative polypeptide; deletion or addition of one or more amino acids atone or more sites in the native polypeptide; or substitution of one ormore amino acids at one or more sites in the native polypeptide. Variantpolypeptides encompassed by the present invention are biologicallyactive, that is they continue to possess the desired biological activityof the native polypeptide, that is, HGGT and/or gamma-tocopherolmethyltransferase and/or 2-methyl-6-phytylbenzoquinol methyltransferaseactivity as described herein. Such variants may result from, forexample, genetic polymorphism or from human manipulation. Biologicallyactive variants of a native HGGT and/or gamma-tocopherolmethyltransferase and/or 2-methyl-6-phytylbenzoquinol methyltransferasepolypeptide of the invention will have at least about 60%, 65%, 70%,generally at least about 75%, 80%, 85%, preferably at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about98%, 99% or more sequence identity to the amino acid sequence for thenative polypeptide as determined by sequence alignment programsdescribed elsewhere herein using default parameters. A biologicallyactive variant of a polypeptide of the invention may differ from thatpolypeptide by as few as 1-15 amino acid residues, as few as 1-10, suchas 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

The polypeptides of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the HGGT and/orgamma-tocopherol methyltransferase and/or 2-methyl-6-phytylbenzoquinolmethyltransferase polypeptides can be prepared by mutations in the DNA.Methods for mutagenesis and nucleotide sequence alterations are wellknown in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci.USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382;U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the polypeptideof interest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be preferred.

Thus, the genes and nucleotide sequences of the invention include boththe naturally occurring sequences as well as mutant forms. Likewise, thepolypeptides of the invention encompass both naturally occurringpolypeptides as well as variations and modified forms thereof. Suchvariants will continue to possess the desired HGGT and/orgamma-tocopherol methyltransferase and/or 2-methyl-6-phytylbenzoquinolmethyltransferase activity. Preferably, the mutations that will be madein the DNA encoding the variant will not place the sequence out ofreading frame and preferably will not create complementary regions thatcould produce secondary mRNA structure. See, EP Patent ApplicationPublication No. 75,444.

The deletions, insertions, and substitutions of the polypeptidesequences encompassed herein are not expected to produce radical changesin the characteristics of the polypeptide. However, when it is difficultto predict the exact effect of the substitution, deletion, or insertionin advance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by routine screening assays. That is, theactivity can be evaluated by assays for HGGT and/or gamma-tocopherolmethyltransferase and/or 2-methyl-6-phytylbenzoquinol methyltransferaseactivity.

Variant nucleotide sequences and polypeptides also encompass sequencesand polypeptides derived from a mutagenic and recombinogenic proceduresuch as DNA shuffling. With such a procedure, one or more different HGGTand/or gamma-tocopherol methyltransferase and/or2-methyl-6-phytylbenzoquinol methyltransferase coding sequences can bemanipulated to create a new HGGT and/or gamma-tocopherolmethyltransferase and/or 2-methyl-6-phytylbenzoquinol methyltransferasepolypeptide possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the HGGTpolynucleotides of the invention and/or other HGGT genes to obtain a newgene coding for a polypeptide with an improved property of interest,such as an increased K_(m) in the case of an enzyme. Likewise, usingthis approach, sequence motifs encoding a domain of interest may beshuffled between the gamma-tocopherol methyltransferase polynucleotidesof the invention and/or other gamma-tocopherol methyltransferase genesto obtain a new gene coding for a polypeptide with an improved propertyof interest, such as an increased K_(m) in the case of an enzyme.Likewise, using this approach, sequence motifs encoding a domain ofinterest may be shuffled between 2-methyl-6-phytylbenzoquinolmethyltransferase polynucleotides of the invention and/or other2-methyl-6-phytylbenzoquinol methyltransferase genes to obtain a newgene coding for a polypeptide with an improved property of interest,such as an increased K_(m) in the case of an enzyme. Strategies for suchDNA shuffling are known in the art. See, for example, Stemmer (1994)Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore etal. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl.Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291;and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other monocots or dicots. In this manner, methods suchas PCR, hybridization, and the like can be used to identify suchsequences based on their sequence homology to the sequences set forthherein. Sequences isolated based on their sequence identity to theentire HGGT and/or gamma-tocopherol methyltransferase and/or2-methyl-6-phytylbenzoquinol methyltransferase nucleotide sequences setforth herein or to fragments thereof are encompassed by the presentinvention. Such sequences include sequences that are orthologs of thedisclosed sequences. By “orthologs” is intended polynucleotides derivedfrom a common ancestral gene and which are found in different species asa result of speciation. Polynucleotides found in different species areconsidered orthologs when their nucleotide sequences and/or theirencoded polypeptide sequences share substantial identity as definedelsewhere herein. Functions of orthologs are often highly conservedamong species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

For clarification, “PCR” or “polymerase chain reaction” is a techniquefor the synthesis of large quantities of specific DNA segments, andconsists of a series of repetitive cycles (Perkin Elmer CetusInstruments, Norwalk, Conn.). Typically, the double stranded DNA is heatdenatured, the two primers complementary to the 3′ boundaries of thetarget segment are annealed at low temperature and then extended at anintermediate temperature. One set of these three consecutive steps isreferred to as a cycle.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the HGGT and/orgamma-tocopherol methyltransferase sequences of the invention. Methodsfor preparation of probes for hybridization and for construction of cDNAand genomic libraries are generally known in the art and are disclosedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2ded., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

For example, an entire HGGT and/or gamma-tocopherol methyltransferaseand/or 2-methyl-6-phytylbenzoquinol methyltransferase sequence disclosedherein, or one or more portions thereof, may be used as a probe capableof specifically hybridizing to corresponding HGGT and/orgamma-tocopherol methyltransferase and/or 2-methyl-6-phytylbenzoquinolmethyltransferase sequences and messenger RNAs. To achieve specifichybridization under a variety of conditions, such probes includesequences that are unique among HGGT and/or gamma-tocopherolmethyltransferase and/or 2-methyl-6-phytylbenzoquinol methyltransferasesequences and are preferably at least about 10 nucleotides in length,and most preferably at least about 20 nucleotides in length. Such probesmay be used to amplify corresponding HGGT and/or gamma-tocopherolmethyltransferase and/or 2-methyl-6-phytylbenzoquinol methyltransferasesequences from a chosen plant by PCR. This technique may be used toisolate additional coding sequences from a desired plant or as adiagnostic assay to determine the presence of coding sequences in aplant. Hybridization techniques include hybridization screening ofplated DNA libraries (either plaques or colonies; see, for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. The duration ofhybridization is generally less than about 24 hours, usually about 4 toabout 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

Isolated sequences that encode a protein with HGGT and/orgamma-tocopherol methyltransferase and/or 2-methyl-6-phytylbenzoquinolmethyltransferase activity and which hybridize under stringentconditions to the HGGT and/or gamma-tocopherol methyltransferase and/or2-methyl-6-phytylbenzoquinol methyltransferase sequences disclosedherein, or to fragments thereof, are encompassed by the presentinvention.

Nucleotides (usually found in their T-monophosphate form) are oftenreferred to herein by their single letter designation as follows: “A”for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” forcytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U”for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y”for pyrimidines (C or T), “K” for G or T, “W” for A or T, “H” for A or Cor T, “D” for A or G or T, “M” for A or C, “S” for C or G, “V” for A orC or G, “B” for C or G or T “I” for inosine, and “N” for A, C, G, or T.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988),supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al. (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990),supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a polypeptide of theinvention. BLAST polypeptide searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a polypeptide of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can beutilized as described in Altschul et al. (1997) Nucleic Acids Res.25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to performan iterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for polypeptides) can be used.Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity using GAP Weight of 50 and LengthWeight of 3; % similarity using Gap Weight of 12 and Length Weight of 4,or any equivalent program. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by the preferred program.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizesthe number of matches and minimizes the number of gaps. GAP considersall possible alignments and gap positions and creates the alignment withthe largest number of matched bases and the fewest gaps. It allows forthe provision of a gap creation penalty and a gap extension penalty inunits of matched bases. GAP must make a profit of gap creation penaltynumber of matches for each gap it inserts. If a gap extension penaltygreater than zero is chosen, GAP must, in addition, make a profit foreach gap inserted of the length of the gap times the gap extensionpenalty. Default gap creation penalty values and gap extension penaltyvalues in Version 10 of the Wisconsin Genetics Software Package forpolypeptide sequences are 8 and 2, respectively. For nucleotidesequences the default gap creation penalty is 50 while the default gapextension penalty is 3. The gap creation and gap extension penalties canbe expressed as an integer selected from the group of integersconsisting of from 0 to 200. Thus, for example, the gap creation and gapextension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

Alternatively, for purposes of the present invention, comparison ofnucleotide or polypeptide sequences for determination of percentsequence identity to the HGGT, gamma-tocopherol methyltransferase or2-methyl-6-phytylbenzoquinol methyltransferase sequences disclosedherein is preferably made using Clustal W found in the MEGALIGN® programof the LASERGENE® bioinformatics computing suite (DNASTAR® Inc.,Madison, Wis.), with the following default parameters. The “defaultparameters” are the parameters pre-set by the manufacturer of theprogram. For amino acid sequence comparisons, default parameters of GapPenalty of 10, a Gap Length Penalty of 0.20, a delay divergent sequenceof 30%, and a DNA Transition Weight of 0.50 are used for multiplealignments; for pairwise alignments the default parameters are GapPenalty of 10.0 and Gap Length of 0.10. Alternatively, amino acidsequence comparisons can be made with Clustal V (described by Higginsand Sharp (1989) CABIOS. 5:151-153) and found in the MEGALIGN® programof the LASERGENE® bioinformatics computing suite (DNASTAR® Inc.,Madison, Wis.). The default parameters of Clustal V for multiplealignments correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10, whilefor pairwise alignments they are KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5. After alignment of the sequences, using the Clustal Vprogram, it is possible to obtain a “percent identity” by viewing the“sequence distances” table on the same program. For nucleotide sequencecomparisons, a Gap Penalty of 10 and Gap Length Penalty of 10 can beused for multiple alignments and a KTUPLE of 2, Gap Penalty of 5, Windowof 4 and Diagonals Saved of 4 can be used for pairwise alignments. Anyequivalent program can also be used to determine percent sequenceidentity. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to polypeptides it is recognizedthat residue positions which are not identical often differ byconservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least90%, and most preferably at least 95%, compared to a reference sequenceusing one of the alignment programs described using standard parameters.One of skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity ofpolypeptides encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 60%, morepreferably at least 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C. lower than theT_(m), depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70% sequenceidentity to a reference sequence, preferably 80%, more preferably 85%,most preferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453. An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

The use of the term “nucleotide constructs” herein is not intended tolimit the present invention to nucleotide constructs comprising DNA.Those of ordinary skill in the art will recognize that nucleotideconstructs, particularly polynucleotides and oligonucleotides, comprisedof ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. Thus, the nucleotide constructs of the present inventionencompass all nucleotide constructs that can be employed in the methodsof the present invention for transforming plants including, but notlimited to, those comprised of deoxyribonucleotides, ribonucleotides,and combinations thereof. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thenucleotide constructs of the invention also encompass all forms ofnucleotide constructs including, but not limited to, single-strandedforms, double-stranded forms, hairpins, stem-and-loop structures, andthe like.

Furthermore, it is recognized that the methods of the invention mayemploy a nucleotide construct that is capable of directing, in atransformed plant, the expression of at least one polypeptide, or atleast one RNA, such as, for example, an antisense RNA that iscomplementary to at least a portion of an mRNA. Typically such anucleotide construct is comprised of a coding sequence for a polypeptideor an RNA operably linked to 5′ and 3′ transcriptional regulatoryregions. Alternatively, it is also recognized that the methods of theinvention may employ a nucleotide construct that is not capable ofdirecting, in a transformed plant, the expression of a polypeptide or anRNA.

In addition, it is recognized that methods of the present invention donot depend on the incorporation of the entire nucleotide construct intothe genome, only that the plant or cell thereof is altered as a resultof the introduction of the nucleotide construct into a cell. In oneembodiment of the invention, the genome may be altered following theintroduction of the nucleotide construct into a cell. For example, thenucleotide construct, or any part thereof, may incorporate into thegenome of the plant. Alterations to the genome of the present inventioninclude, but are not limited to, additions, deletions, and substitutionsof nucleotides in the genome. While the methods of the present inventiondo not depend on additions, deletions, or substitutions of anyparticular number of nucleotides, it is recognized that such additions,deletions, or substitutions comprise at least one nucleotide.

The nucleotide constructs of the invention also encompass nucleotideconstructs that may be employed in methods for altering or mutating agenomic nucleotide sequence in an organism, including, but not limitedto, homologous recombination, chimeric vectors, chimeric mutationalvectors, chimeric repair vectors, mixed-duplex oligonucleotides,self-complementary chimeric oligonucleotides, and recombinogenicoligonucleobases. See, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325;5,760,012; 5,795,972; and 5,871,984; all of which are hereinincorporated by reference. See also, PCT Publication No. WO 98/49350,PCT Publication No. WO 99/07865, PCT Publication No. WO 99/25821, PCTPublication No. WO03093428, Jeske et al. (2001) EMBO 20:6158-6167, andBeetham et al. (1999) Proc. Natl. Acad. Sci. USA 96:8774-8778; hereinincorporated by reference.

The HGGT, gamma-tocopherol methyltransferase and2-methyl-6-phytylbenzoquinol methyltransferase sequences of theinvention are provided in expression cassettes for expression in theplant of interest. The cassette(s) will include at least one 5′ and 3′regulatory sequences operably linked to a HGGT and/or gamma-tocopherolmethyltransferase and/or 2-methyl-6-phytylbenzoquinol methyltransferasenucleotide sequence of the invention. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two polypeptide coding regions, contiguousand in the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the HGGT and/or gamma-tocopherolmethyltransferase and/or 2-methyl-6-phytylbenzoquinol methyltransferasenucleotide sequence to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containselectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aHGGT and/or gamma-tocopherol methyltransferase and/or2-methyl-6-phytylbenzoquinol methyltransferase polynucleotide sequenceof the invention, and a transcriptional and translational terminationregion functional in plants. These genes can be added either alone or incombination. The transcriptional initiation region, the promoter, may benative or analogous or foreign or heterologous to the plant host.Additionally, the promoter may be the natural sequence or alternativelya synthetic sequence. By “foreign” is intended that the transcriptionalinitiation region is not found in the native plant into which thetranscriptional initiation region is introduced. As used herein, achimeric gene comprises a coding sequence operably linked to atranscription initiation region that is heterologous to the codingsequence.

While it may be preferable to express the sequences using heterologouspromoters, the native promoter sequences may be used. Such constructswould change expression levels of HGGT and/or gamma-tocopherolmethyltransferase and/or 2-methyl-6-phytylbenzoquinol methyltransferasein the plant, plant cell or other host. Thus, the phenotype of theplant, plant cell or other host is altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,or may be derived from another source. Convenient termination regionsare available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See alsoGuerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991)Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen etal. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.(1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed plant. That is, the genes can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, U.S. Pat. Nos.5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology154:9-20), and human immunoglobulin heavy-chain binding protein (BiP)(Macejak et al. (1991) Nature 353:90-94); untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie etal. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp.237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al.(1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, chemically regulated,tissue-preferred, or other promoters for expression in plants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in PCTPublication No. WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin(McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen etal. (1989) Plant Mot. Biol. 12:619-632 and Christensen et al. (1992)Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl.Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. Pat. No. 5,659,026), and the like. Other constitutivepromoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and6,177,611.

Chemically regulated promoters can be used to modulate the expression ofa gene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be a chemicalinducible promoter, where application of the chemical induces geneexpression, or a chemical repressible promoter, where application of thechemical represses gene expression. Chemical inducible promoters areknown in the art and include, but are not limited to, the maize In2-2promoter, which is activated by benzenesulfonamide herbicide safeners,the maize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides, and the tobaccoPR-1a promoter, which is activated by salicylic acid. Other chemicallyregulated promoters of interest include steroid-responsive promoters(see, for example, the glucocorticoid-inducible promoter in Schena etal. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al.(1998) Plant J. 14(2):247-257) and tetracycline-inducible andtetracycline-repressible promoters (see, for example, Gatz et al. (1991)Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced HGGTand/or gamma-tocopherol methyltransferase and/or2-methyl-6-phytylbenzoquinol methyltransferase expression within aparticular plant tissue. Tissue-preferred promoters include Yamamoto etal. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant CellPhysiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen. Genet.254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al.(1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) PlantPhysiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2): 207-218 (soybean root-preferred glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-preferredcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-preferred promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-preferred promoters isolated fromhemoglobin genes from the nitrogen-fixing nonlegume Parasponiaandersonii and the related non-nitrogen-fixing nonlegume Trema tomentosaare described. The promoters of these genes were linked to aβ-glucuronidase reporter gene and introduced into both the nonlegumeNicotiana tabacum and the legume Lotus corniculatus, and in bothinstances root-preferred promoter activity was preserved. Leach andAoyagi (1991) describe their analysis of the promoters of the highlyexpressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes(see Plant Science (Limerick) 79(1):69-76). They concluded that enhancerand tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri et al. (1989) used gene fusion to lacZ to show that theAgrobacterium T-DNA gene encoding octopine synthase is especially activein the epidermis of the root tip and that the TR2′ gene is rootpreferred in the intact plant and stimulated by wounding in leaf tissue,an especially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TR1′gene, fused to nptII (neomycin phosphotransferase II) showed similarcharacteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol.29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol.25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363;5,459,252; 5,401,836; 5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529; hereinincorporated by reference). Gamma-zein is an endosperm-specificpromoter. Globulin 1 (Glb-1) is a representative embryo-specificpromoter. For dicots, seed-specific promoters include, but are notlimited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin, and the like. For monocots, seed-specific promoters include,but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein,gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, etc. See also thepromoters found in the following: End1 and End2 (WO 00/12733), Lec1 (WO2002/42424), Jip1 (WO 2002/42424), EAP1 (U.S. Patent Publication No.2004/0210043), ODP2 (U.S. Patent Publication No. 2005/0223432); all ofwhich are herein incorporated by reference.

In one embodiment, the nucleic acids of interest are targeted to thechloroplast for expression. In this manner, where the nucleic acid ofinterest is not directly inserted into the chloroplast, the expressioncassette will additionally contain a nucleic acid encoding a transitpeptide to direct the gene product of interest to the chloroplasts orother plastids. Such transit peptides are known in the art. See, forexample, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clarket al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987)Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res.Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.

The HGGT and/or gamma-tocopherol methyltransferase and/or2-methyl-6-phytylbenzoquinol methyltransferase polypeptides of theinvention can be targeted to specific compartments within the plantcell. Methods for targeting polypeptides to a specific compartment areknown in the art. Generally, such methods involve modifying thenucleotide sequence encoding the polypeptide in such a manner as to addor remove specific amino acids from the polypeptide encoded thereby.Such amino acids comprise targeting signals for targeting thepolypeptide to a specific compartment such as, for example, a theplastid, the nucleus, the endoplasmic reticulum, the vacuole, themitochondrion, the peroxisome, the Golgi apparatus, and for secretionfrom the cell. Targeting sequences for targeting a polypeptide to aspecific cellular compartment, or for secretion, are known to those ofordinary skill in the art. Chloroplast-targeting or plastid-targetingsequences are known in the art and include the chloroplast small subunitof ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro SilvaFilho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991)J. Biol. Chem. 266(5):3335-3342); 5-(enolpyruvyl)shikimate-3-phosphatesynthase (EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb.22(6):789-810); tryptophan synthase (Zhao et al. (1995) J. Biol. Chem.270(11):6081-6087); plastocyanin (Lawrence et al. (1997) J. Biol. Chem.272(33):20357-20363); chorismate synthase (Schmidt et al. (1993) J.Biol. Chem. 268(36):27447-27457); and the light harvesting chlorophylla/b binding protein (LHBP) (Lamppa et al. (1988) J. Biol. Chem.263:14996-14999). See also Von Heijne et al. (1991) Plant Mol. Biol.Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550;Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al.(1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al.(1986) Science 233:478-481.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin Bphosphotransferase, as well as genes conferring resistance to herbicidalcompounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992)Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff(1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724. Such disclosures are herein incorporatedby reference.

Other genes that could serve as selectable or scorable markers in therecovery of transgenic events but that might not be required in thefinal product would include, but are not limited to: GUS(β-glucoronidase), Jefferson (1987) Plant Mol. Biol. Rep. 5:387);fluorescent proteins, such as, GFP (green florescence protein), YFP(yello florescence protein), RFP (red florescence protein) and CYP (cyanflorescence protein), WO 00/34321, WO 00/34526, WO 00/34323, WO00/34322, WO 00/34318, WO 00/34319, WO 00/34320, WO 00/34325, WO00/34326, WO 00/34324, Chalfie et al. (1994) Science 263:802;luciferase, Teeri et al. (1989) EMBO J. 8:343; and the maize genesencoding for anthocyanin production, Ludwig et al. (1990) Science247:449.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

The invention involves transforming host cells with the nucleotideconstructs of the invention. Generally, the nucleotide construct willcomprise a HGGT nucleotide and/or gamma-tocopherol methyltransferaseand/or 2-methyl-6-phytylbenzoquinol methyltransferase sequence of theinvention, either a full length sequence or functional fragment thereof,operably linked to a promoter that drives expression in the host cell ofinterest. Host cells include, but are not limited to: plant cells;animal cells; fungal cells, particularly yeast cells; and bacterialcells.

The methods of the invention involve introducing a nucleotide constructinto a plant. By “introducing” is intended presenting to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notdepend on a particular method for introducing a nucleotide construct toa plant, only that the nucleotide construct gains access to the interiorof at least one cell of the plant. Methods for introducing nucleotideconstructs into plants are known in the art including, but not limitedto, stable transformation methods, transient transformation methods, andvirus-mediated methods.

By “stable transformation” is intended that the nucleotide constructintroduced into a plant integrates into the genome of the plant and iscapable of being inherited by progeny thereof. By “transienttransformation” is intended that a nucleotide construct introduced intoa plant does not integrate into the genome of the plant.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particleacceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050;Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No.5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe etal. (1988) Biotechnology 6:923-926); and Lec1 transformation (PCTPublication No. 00/028058). Also see Weissinger et al. (1988) Ann. Rev.Genet. 22:421-477; Sanford et al. (1987) Particulate Science andTechnology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926(soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York),pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

The nucleotide constructs of the invention may also be introduced intoplants by contacting plants with a virus or viral nucleic acids.Generally, such methods involve incorporating a nucleotide construct ofthe invention within a viral DNA or RNA molecule. It is recognized thata HGGT and/or gamma-tocopherol methyltransferase and/or2-methyl-6-phytylbenzoquinol methyltransferase of the invention may beinitially synthesized as part of a viral polyprotein, which later may beprocessed by proteolysis in vivo or in vitro to produce the desiredrecombinant polypeptide. Further, it is recognized that promoters of theinvention also encompass promoters utilized for transcription by viralRNA polymerases. Methods for introducing nucleotide constructs intoplants and expressing a polypeptide encoded therein, involving viral DNAor RNA molecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931; hereinincorporated by reference.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

The nucleic acids of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.

As used herein, “transformed plants” include those plants directlytransformed as provided herein, as well as plants that have the directlytransformed plants in their pedigree and retain the change in genotype,such as the inclusion of the expression cassette, created by theoriginal transformation. The terms “transformed plants” and “transgenicplants” are used interchangeably herein.

The present invention may be used for transformation of any plantspecies, including, but not limited to, maize (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the present invention include, for example, pinessuch as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), andMonterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii);Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Preferably, plants of the present invention are crop plants (forexample, maize, alfalfa, sunflower, Brassica, soybean, cotton,safflower, peanut, sorghum, wheat, barley, rice, sorghum, rye, millet,tobacco, etc.), more preferably cereal plants, yet more preferablymaize, wheat, barley, rice, sorghum, rye and millet plants.

In some embodiments, the activity of a gene of the invention is reducedor eliminated by transforming a plant cell with an expression cassetteexpressing a polynucleotide that inhibits the expression of a targetgene. The polynucleotide may inhibit the expression of one or moretarget genes directly, by preventing translation of the target genemessenger RNA, or indirectly, by encoding a polypeptide that inhibitsthe transcription or translation of a gene encoding the target gene.Methods for inhibiting or eliminating the expression of a gene in aplant are well known in the art, and any such method may be used in thepresent invention to inhibit the expression of one or more plant genes,such as, HGGT and/or gamma-tocoperol methyltransferase and/or2-methyl-6-phytylbenzoquinol methyltransferase.

In accordance with the present invention, the expression of a targetgene protein is inhibited if the protein level of the target gene isstatistically lower than the protein level of the same target gene in aplant that has not been genetically modified or mutagenized to inhibitthe expression of that target gene. In particular embodiments of theinvention, the protein level of the target gene in a modified plantaccording to the invention is less than 95%, less than 90%, less than85%, less than 80%, less than 75%, less than 70%, less than 65%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10%, or less than 5% of the protein level of the same targetgene in a plant that is not a mutant or that has not been geneticallymodified to inhibit the expression of that target gene. The expressionlevel of the target gene may be measured directly, for example, byassaying for the level of target gene expressed in the maize cell orplant, or indirectly, for example, by measuring the activity of thetarget gene enzyme in the maize cell or plant. The activity of a targetgene protein is “eliminated” according to the invention when it is notdetectable by at least one assay method described elsewhere herein.

Many methods may be used to reduce or eliminate the activity of a targetgene. More than one method may be used to reduce the activity of asingle target gene. In addition, combinations of methods may be employedto reduce or eliminate the activity of two or more different targetgenes. Non-limiting examples of methods of reducing or eliminating theexpression of a plant target are given below.

Many techniques for gene silencing are well known to one of skill in theart, including but not limited to knock-outs, such as, by insertion of atransposable element such as Mu (Vicki Chandler, The Maize Handbook ch.118 (Springer-Verlag 1994)), other genetic elements such as a FRT, Loxor other site specific integration site, alteration of the target geneby homologous recombination (Bolon, B. Basic Clin. Pharmacol. Toxicol.95:4,12,154-61 (2004); Matsuda and Alba, A., Methods Mol. Bio.259:379-90 (2004); Forlino, et. al., J. Biol. Chem. 274:53, 37923-30(1999), antisense technology (see, e.g., Sheehy et al. (1988) PNAS USA85:8805-8809; and U.S. Pat. Nos. 5,107,065; 5,453,566; and 5,759,829;U.S. Patent Publication No. 20020048814); sense suppression (e.g., U.S.Pat. No. 5,942,657; Flavell et al. (1994) Proc. Natl. Acad. Sci. USA 91:3490-3496; Jorgensen et al. (1996) Plant Mol. Biol. 31: 957-973;Johansen and Carrington (2001) Plant Physiol. 126: 930-938; Broin et al.(2002) Plant Cell 14: 1417-1432; Stoutjesdijk et al (2002) PlantPhysiol. 129: 1723-1731; Yu et al. (2003) Phytochemistry 63: 753-763;and U.S. Pat. Nos. 5,034,323, 5,283,184, and 5,942,657; U.S. PatentPublication No. 20020048814); RNA interference (Napoli et al. (1990)Plant Cell 2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev.13:139-141; Zamore et al. (2000) Cell 101:25-33; and Montgomery et al.(1998) PNAS USA 95:15502-15507), virus-induced gene silencing (Burton,et al. (2000) Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op.Plant Bio. 2:109-113); target-RNA-specific ribozymes (Haseloff et al.,(1988) Nature 334: 585-591, U.S. Pat. No. 4,987,071); hairpin structures(Smith et al. (2000) Nature 407:319-320; Waterhouse et al. (1998) Proc.Natl. Acad. Sci. USA 95: 13959-13964, Liu et al. (2002) Plant Physiol.129: 1732-1743, Waterhouse and Helliwell (2003) Nat. Rev. Genet.4:29-38; Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk et al. (2002) Plant Physiol. 129: 1723-1731;Panstruga et al. (2003) Mol. Biol. Rep. 30: 135-140; Smith et al. (2000)Nature 407: 319-320; Smith et al. (2000) Nature 407:319-320; Wesley etal. (2001) Plant J. 27: 581-590; Wang and Waterhouse (2001) Curr. Opin.Plant Biol. 5: 146-150; Waterhouse and Helliwell (2003) Nat. Rev. Genet.4: 29-38; Helliwell and Waterhouse (2003) Methods 30: 289-295;Pandolfini et al. BMC Biotechnology 3:7; U.S. Patent Publication No.20030180945; U.S. Patent Publication No. 20030175965; WO 99/49029; WO99/53050; WO 99/61631; and WO 00/49035); transcriptional gene silencing(TGS) (Aufsatz et al. (2002) Proc. Nat'l. Acad. Sci. 99 (Suppl.4):16499-16506; Mette et al. (2000) EMBO J. 19(19):5194-5201; microRNA(Aukerman & Sakai (2003) Plant Cell 15:2730-2741); ribozymes (Steineckeet al. (1992) EMBO J. 11:1525; and Perriman et al. (1993) Antisense Res.Dev. 3:253); oligonucleotide mediated targeted modification (e.g., WO03/076574 and WO 99/25853); Zn-finger targeted molecules (e.g., WO01/52620; WO 03/048345; and WO 00/42219); methods of using amplicons(Angell and Baulcombe (1997) EMBO J. 16: 3675-3684, Angell and Baulcombe(1999) Plant J. 20: 357-362, and U.S. Pat. No. 6,646,805);polynucleotides that encode an antibody that binds to protein ofinterest (Conrad and Sonnewald (2003) Nature Biotech. 21: 35-36);transposon tagging (Maes et al. (1999) Trends Plant Sci. 4: 90-96;Dharmapuri and Sonti (1999) FEMS Microbiol. Lett. 179: 53-59; Meissneret al. (2000) Plant J. 22: 265-274; Phogat et al. (2000) J. Biosci. 25:57-63; Walbot (2000) Curr. Opin. Plant Biol. 2: 103-107; Gai et al.(2000) Nucleic Acids Res. 28: 94-96; Fitzmaurice et al. (1999) Genetics153: 1919-1928; the TUSC process for selecting Mu insertions in selectedgenes (Bensen et al. (1995) Plant Cell 7: 75-84; Mena et al. (1996)Science 274: 1537-1540; and U.S. Pat. No. 5,962,764); other forms ofmutagenesis, such as ethyl methanesulfonate-induced mutagenesis,deletion mutagenesis, and fast neutron deletion mutagenesis used in areverse genetics sense (with PCR) to identify plant lines in which theendogenous gene has been deleted (Ohshima et al. (1998) Virology 243:472-481; Okubara et al. (1994) Genetics 137: 867-874; and Quesada et al.(2000) Genetics 154: 421-436; TILLING (Targeting Induced Local LesionsIn Genomes) (McCallum et al. (2000) Nat. Biotechnol. 18: 455-457) andother methods or combinations of the above methods known to those ofskill in the art. Each reference is herein incorporated by reference

An expression cassette is designed to reduce activity of the target genemay express an RNA molecule corresponding to all or part of a messengerRNA encoding a target gene in the sense or antisense orientation or acombination of both sense and antisense. Overexpression of the RNAmolecule can result in reduced expression of the native gene.Accordingly, multiple plant lines transformed with the sense suppressionexpression cassette are screened to identify those that show thegreatest inhibition of the target gene's expression.

The polynucleotide used for target gene suppression may correspond toall or part of the sequence encoding the target gene, all or part of the5′ and/or 3′ untranslated region of a target gene transcript, or all orpart of both the coding region and the untranslated regions of atranscript encoding of the target gene or all or part of the promotersequence responsible for expression of the target gene. A polynucleotideused for sense suppression or other gene silencing methods may share99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 85%,80%, or less sequence identity with the target sequence. When portionsof the polynucleotides are used to disrupt the expression of the targetgene, generally, sequences of at least 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 900nucleotides or 1 kb or greater may be used.

The present invention includes:

In one embodiment, a transformed plant comprising in its genome: (a) afirst recombinant nucleic acid molecule comprising at least oneregulatory sequence operably linked to at least one nucleotide sequenceselected from the group consisting of: (i) a nucleotide sequenceencoding a polypeptide having gamma-tocopherol methyltransferaseactivity; (ii) a nucleotide sequence set forth in SEQ ID NOs: 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39; (iii) a nucleotidesequence encoding the amino acid sequence set forth in SEQ ID NOs: 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40; (iv) anucleotide sequence having at least 80% sequence identity to thenucleotide sequence set forth in any one of (i)-(iii), wherein thenucleotide sequence encodes a polypeptide having gamma-tocopherolmethyltransferase activity; and (v) a nucleotide sequence that is fullycomplementary to the nucleotide sequence of any one of (i)-(iv); (b) asecond recombinant nucleic acid molecule comprising at least oneregulatory sequence operably linked to at least one nucleotide sequenceselected from the group consisting of: (vi) a nucleotide sequenceencoding a polypeptide having homogentisate geranylgeranyl transferaseactivity; (vii) a nucleotide sequence set forth in SEQ ID NOs: 1, 3, 5,7, or 9; (viii) a nucleotide sequence encoding the amino acid sequenceset forth in SEQ ID NOs: 2, 4, 6, 8, or 10; (ix) a nucleotide sequencehaving at least 80% sequence identity to the nucleotide sequence setforth in any one of (vi)-(viii), wherein the nucleotide sequence encodesa polypeptide having homogentisate geranylgeranyl transferase activity;and (x) a nucleotide sequence that is fully complementary to thenucleotide sequence of any one of (vi)-(ix); and (c) a third recombinantnucleic acid molecule comprising at least one regulatory sequenceoperably linked to at least one nucleotide sequence selected from thegroup consisting of: (xi) a nucleotide sequence encoding a polypeptidehaving 2-methyl-6-phytylbenzoquinol methyltransferase activity; (xii) anucleotide sequence set forth in SEQ ID NO:53; (xiii) a nucleotidesequence encoding the amino acid sequence set forth in SEQ ID NOs: 54,61, 62, 63, 64, 65, 66, 67, 68, 69 or 70; (xiv) a nucleotide sequencehaving at least 80% sequence identity to the nucleotide sequence setforth in any one of (xi)-(xii), wherein the nucleotide sequence encodesa polypeptide having 2-methyl-6-phytylbenzoquinol methyltransferaseactivity; (xv) a nucleotide sequence encoding a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity, wherein theamino acid sequence of the polypeptide has at least 95% sequenceidentity to the amino acid sequence set forth in SEQ ID NOs: 54, 61, 62,63, 64, 65, 66, 67, 68, 69 or 70; and (xvi) a nucleotide sequence thatis fully complementary to the nucleotide sequence of any one of(xi)-(xv); wherein the first recombinant nucleic acid molecule, thesecond recombinant nucleic acid molecule and the third recombinantnucleic acid molecule are stably incorporated into the genome of thetransformed plant.

In another embodiment, a transformed plant comprising in its genome atleast one recombinant nucleic acid molecule seclected from the groupconsisting of the first recombinant nucleic acid molecule, the secondrecombinant nucleic acid molecule and the third recombinant nucleic acidmolecule of above, wherein the at least one recombinant nucleic acidmolecule is stably incorporated into the genome of the transformedplant.

In another embodiment, the transformed plant may be a monocot selectedfrom the group consisting of maize, wheat, rice, sorghum, barley, milletand rye.

In another embodiment, the transformed plant may be a dicot selectedfrom the group consisting of soybean, Brassica sp., alfalfa, safflower,sunflower, cotton, peanut, canola, Arabidopsis, tobacco and potato.

In another embodiment, the at least one regulatory sequence of thefirst, second or third recombinant nucleic acid molecule comprises atleast one promoter selected from the group consisting of seed-preferred,constitutive, chemically regulated, tissue-preferred, anddevelopmentally regulated promoters.

In another embodiment, the invention includes seed of the transformedplant, wherein said seed comprises in its genome the first recombinantnucleic acid molecule, the second recombinant nucleic acid molecule andthe third recombinant nucleic acid molecule.

In another embodiment, the transformed plant of the invention produces aseed with an increased level of alpha-tocotrienol, beta-tocotrienol, orboth, relative to a plant with a similar genetic background but lackingsaid first recombinant nucleic acid molecule, said second recombinantnucleic acid molecule and said third recombinant nucleic acid molecule.

In another embodiment, the invention includes a method of increasing thelevel of alpha-tocotrienol, beta-tocotrienol, or both, in a plant,comprising stably incorporating into a plant genome: (a) a firstrecombinant nucleic acid molecule comprising at least one regulatorysequence operably linked to at least one nucleotide sequence selectedfrom the group consisting of: (i) a nucleotide sequence encoding apolypeptide having gamma-tocopherol methyltransferase activity; (ii) anucleotide sequence set forth in SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37 or 39; (iii) a nucleotide sequence encodingthe amino acid sequence set forth in SEQ ID NOs: 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38 or 40; (iv) a nucleotide sequence havingat least 80% sequence identity to the nucleotide sequence set forth inany one of (i)-(iii), wherein the nucleotide sequence encodes apolypeptide having gamma-tocopherol methyltransferase activity; and (v)a nucleotide sequence that is complementary to the nucleotide sequenceof any one of (i)-(iv); (b) a second recombinant nucleic acid moleculecomprising at least one regulatory sequence operably linked to at leastone nucleotide sequence selected from the group consisting of: (vi) anucleotide sequence encoding a polypeptide having homogentisategeranylgeranyl transferase activity; (vii) a nucleotide sequence setforth in SEQ ID NOs: 1, 3, 5, 7, or 9; (viii) a nucleotide sequenceencoding the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, or10; (ix) a nucleotide sequence having at least 80% sequence identity tothe nucleotide sequence set forth in any one of (vi)-(viii), wherein thenucleotide sequence encodes a polypeptide having homogentisategeranylgeranyl transferase activity; and (x) a nucleotide sequence thatis complementary to the nucleotide sequence of any one of (vi)-(ix); and(c) a third recombinant nucleic acid molecule comprising at least oneregulatory sequence operably linked to at least one nucleotide sequenceselected from the group consisting of: (xi) a nucleotide sequenceencoding a polypeptide having 2-methyl-6-phytylbenzoquinolmethyltransferase activity; (xii) a nucleotide sequence set forth in SEQID NO:53; (xiii) a nucleotide sequence encoding the amino acid sequenceset forth in SEQ ID NOs: 54, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70;(xiv) a nucleotide sequence having at least 80% sequence identity to thenucleotide sequence set forth in any one of (xi)-(xii), wherein thenucleotide sequence encodes a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity; (xv) anucleotide sequence encoding a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity, wherein theamino acid sequence of the polypeptide has at least 95% sequenceidentity to the amino acid sequence set forth in SEQ ID NOs: 54, 61, 62,63, 64, 65, 66, 67, 68, 69 or 70; and (xvi) a nucleotide sequence thatis fully complementary to the nucleotide sequence of any one of(xi)-(xv); and selecting a transformed plant that has an increased levelof alpha-tocotrienol, beta-tocotrienol, or both, relative to a plantwith a similar genetic background but lacking said first recombinantnucleic acid molecule, said second recombinant nucleic acid molecule andsaid third recombinant nucleic acid molecule.

In another embodiment, a method in which the first, second and thirdrecombinant nucleic acid molecules are incorporated into the plantgenome by co-transformation of a plant cell.

In another embodiment, a method in which at least one of said first,second or third recombinant nucleic acid molecules is incorporated intothe plant genome by re-transformation of a transformed plant cell,wherein said transformed plant cell comprises at least one of saidfirst, second or third recombinant nucleic acid molecules.

In another embodiment, a method in which at least one of said first,second or third recombinant nucleic acid molecule is incorporated intothe plant genome by breeding.

In another embodiment, a method in which the at least one regulatorysequence of the first, second or third recombinant nucleic acid moleculecomprises at least one promoter selected from the group consisting ofseed-preferred, constitutive, chemically regulated, tissue-preferred,and developmentally regulated promoters.

In another embodiment, the invention includes methods for transformingplants and plants cells to change the oil content therein comprisingtransforming a plant with one to three nucleotide sequences alone or inany combination of two or three nucleotide sequences. The methodcomprises; (a) obtaining a first plant comprising in its genome a firstrecombinant nucleic acid molecule comprising at least one regulatorysequence operably linked to a nucleotide sequence encoding a polypeptidehaving gamma-tocopherol methyltransferase activity; (b) crossing thetransgenic plant of step (a) with a second plant comprising in itsgenome a second recombinant nucleic acid molecule comprising at leastone regulatory sequence operably linked to a nucleotide sequenceencoding a polypeptide having homogentisate geranylgeranyl transferaseactivity; (c) crossing the transgenic plant of step (b) with a thirdplant comprising in its genome a third recombinant nucleic acid moleculecomprising at least one regulatory sequence operably linked to anucleotide sequence encoding a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity; and (d)obtaining a progeny plant from said step (c) crossing, wherein saidprogeny plant comprises in its genome the first recombinant nucleic acidmolecule, the second recombinant nucleic acid molecule and the thirdrecombinant nucleic acid molecule, and wherein said progeny plantexhibits an increased level of alpha-tocotrienol, beta-tocotrienol, orboth, relative to a plant with a similar genetic background but lackingsaid first recombinant nucleic acid molecule, said second recombinantnucleic acid molecule and said third recombinant nucleic acid molecule.

In another embodiment, a method of increasing the level ofalpha-tocotrienol, beta-tocotrienol, or both, in a plant, comprising:(a) obtaining a first transformed plant comprising in its genome a firstrecombinant nucleic acid molecule comprising at least one regulatorysequence operably linked to at least one nucleotide sequence selectedfrom the group consisting of: (i) a nucleotide sequence encoding apolypeptide having gamma-tocopherol methyltransferase activity; (ii) anucleotide sequence set forth in SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37 or 39; (iii) a nucleotide sequence encodingthe amino acid sequence set forth in SEQ ID NOs: 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38 or 40; (iv) a nucleotide sequence havingat least 80% sequence identity to the entire coding sequence of thenucleotide sequence set forth in any one or (i)-(iii), wherein thenucleotide sequence encodes a polypeptide having gamma-tocopherolmethyltransferase activity; and (v) a nucleotide sequence that iscomplementary to the nucleotide sequence of any one of (i)-(iv); (b)crossing the transformed plant of step (a) with a second transformedplant comprising within its genome a second recombinant nucleic acidmolecule comprising at least one regulatory sequence operably linked toat least one nucleotide sequence selected from the group consisting of:(vi) a nucleotide sequence encoding a polypeptide having homogentisategeranylgeranyl transferase activity; (vii) a nucleotide sequence setforth in SEQ ID NOs: 1, 3, 5, 7 or 9; (viii) a nucleotide sequenceencoding the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8 or10; (ix) a nucleotide sequence having at least 80% sequence identity tothe entire coding sequence of the nucleotide sequence set forth in(vi)-(viii), wherein the nucleotide sequence encodes a polypeptidehaving homogentisate geranylgeranyl transferase activity; and (x) anucleotide sequence that is complementary to the nucleotide sequence ofany one of (vi)-(ix); and (c) crossing the transformed plant of step (b)with a third transformed plant comprising within its genome a thirdrecombinant nucleic acid molecule comprising at least one regulatorysequence operably linked to at least one nucleotide sequence selectedfrom the group consisting of: (xi) a nucleotide sequence encoding apolypeptide having 2-methyl-6-phytylbenzoquinol methyltransferaseactivity; (xii) a nucleotide sequence set forth in SEQ ID NO:53; (xiii)a nucleotide sequence encoding the amino acid sequence set forth in SEQID NOs: 54 or 70; (xiv) a nucleotide sequence having at least 80%sequence identity to the nucleotide sequence set forth in any one of(xi)-(xii), wherein the nucleotide sequence encodes a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity; (xv) anucleotide sequence encoding a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity, wherein theamino acid sequence of the polypeptide has at least 95% sequenceidentity to the amino acid sequence set forth in SEQ ID NOs: 54, 61, 62,63, 64, 65, 66, 67, 68, 69 or 70; and (xvi) a nucleotide sequence thatis fully complementary to the nucleotide sequence of any one of(xi)-(xv); and selecting a progeny plant from said step (c) crossing,wherein said progeny plant comprises in its genome the first recombinantnucleic acid molecule, the second recombinant nucleic acid molecule andthe third recombinant nucleic acid molecule, and wherein said progenyplant exhibits an increased level of alpha-tocotrienol,beta-tocotrienol, or both, relative to a plant with a similar geneticbackground but lacking said first recombinant nucleic acid molecule,said second recombinant nucleic acid molecule and said third recombinantnucleic acid molecule.

In another embodiment, any of the methods of the invention wherein theplant is a monocot is selected from the group consisting of maize,wheat, rice, sorghum, barley, millet and rye.

In another embodiment, any of the methods of the invention wherein theplant is a dicot is selected from the group consisting of soybean,Brassica sp., alfalfa, safflower, sunflower, cotton, peanut, canola,Arabidopsis, tobacco and potato.

In another embodiment, the invention includes transformed seed orbyproducts of any of the transformed plants of the invention.

In another embodiment, the transformed seed of the invention has analpha-tocotrienol level of at least 20 ppm.

In another embodiment, the transformed seed of the invention containsalpha-tocotrienol in an amount of at least 20% of total tocopherol andtocotrienol content in the transformed seed.

In another embodiment, the transformed seed of the invention has analpha-tocotrienol content of at least 70% of total combined tocopheroland tocotrienol content in the transformed seed.

In another embodiment, the transformed seed of the invention contains acombined level of alpha-tocotrienol and alpha-tocopherol of at least 95%of total tocopherol and tocotrienol content in the transformed seed.

In another embodiment, a method of improving the tissue quality of ananimal, comprising feeding the animal the transformed seed of theinvention.

In another embodiment, the tissue is meat and the quality of the meat ismeasured by at least one criteria selected from the group consisting ofincreased pH, improved food color, improved oxidative stability,increased shelf life and reduced purge.

In another embodiment, the animal is a ruminant, preferably cattle.

In another embodiment, the animal is a non-ruminant, preferably swine orpoultry.

In another embodiment, an isolated polynucleotide comprising SEQ IDNO:57.

In another embodiment, an isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity, wherein thepolypeptide has an amino acid sequence of at least 95% sequenceidentity, based on the Clustal W method of alignment, when compared toSEQ ID NO:54 or 70, or (b) a complement of the nucleotide sequence,wherein the complement and the nucleotide sequence consist of the samenumber of nucleotides and are 100% complementary. The amino acidsequence of the polypeptide preferably comprises SEQ ID NO:54 or 70. Thenucleotide sequence preferably comprises SEQ ID NO:53.

In another embodiment, the present invention includes a vectorcomprising any of the isolated polynucleotides of the present invention.

In another embodiment, the present invention includes a recombinant DNAconstruct comprising any of the isolated polynucleotides of the presentinvention operably linked to at least one regulatory sequence.

In another embodiment, the present invention concerns a method fortransforming a cell comprising transforming a cell with any of theisolated polynucleotides of the present invention. The cell transformedby this method is also included. In particular embodiments, the cell iseukaryotic cell, e.g., a yeast, insect or plant cell, or prokaryotic,e.g., a bacterium.

In another embodiment, the present invention includes a method forproducing a transgenic plant comprising transforming a plant cell withany of the isolated polynucleotides or recombinant DNA constructs of thepresent invention and regenerating a transgenic plant from thetransformed plant cell. The invention is also directed to the transgenicplant produced by this method, and transgenic seed obtained from thistransgenic plant.

In another embodiment, an isolated polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity, wherein thepolypeptide has an amino acid sequence of at least 95% sequenceidentity, based on the Clustal W method of alignment, when compared toone of SEQ ID NO:54 or 70. The amino acid sequence of the polypeptideprefereably comprises SEQ ID NO:54 or 70.

In another embodiment, a method for isolating a polypeptide having2-methyl-6-phytylbenzoquinol methyltransferase activity comprisingisolating the polypeptide from a cell or culture medium of the cell,wherein the cell comprises a recombinant DNA construct comprising thepolynucleotide of the invention operably linked to at least oneregulatory sequence.

In another embodiment, a method of altering the level of expression of a2-methyl-6-phytylbenzoquinol methyltransferase in a host cellcomprising: (a) transforming a host cell with the recombinant DNAconstruct of the invention; and (b) growing the transformed host cellunder conditions that are suitable for expression of the recombinant DNAconstruct wherein expression of the recombinant DNA construct results inproduction of altered levels of the 2-methyl-6-phytylbenzoquinolmethyltransferase in the transformed host cell.

The following examples are presented by way of illustration, not by wayof limitation.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration, not by way of limitation. From the above discussionand these Examples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Thus, variousmodifications of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

Example 1 Alpha- and Beta-Tocotrienol Production in Arabidopsis thalianaby Transgenic Expression of Barley HGGT and Soybean Gamma-TocopherolMethyltransferase

The cDNA for barley homogentisate geranylgeranyl transferase (HGGT)(bdl2c.pk006.o2; SEQ ID NO:2) and soybean gamma-tocopherolmethyltransferase (sah1c.pk004.g2; SEQ ID NO:12) were expressed inArabidopsis thaliana to demonstrate the feasability of these cDNA foralpha and beta-tocotrienol production in transgenic plants.

A transformation vector was constructed using standard molecular toolsthat expressed the barley HGGT gene under the control of theβ-conglycinin promoter of soybean (Beachy et al., EMBO J. 4:3047-3053(1985)) and the soybean gamma-tocopherol methyltransferase gene underthe control of the Kti promoter (Kunitz Trypsin Inhibitor, Jofuku et.al., (1989) Plant Cell 1:1079-1093).

The 1.1 kb DNA fragment containing the soybean gamma-tocopherolmethyltransferase gene was excised from SC1 (see Example 3) using therestriction enzyme NotI, and ligated, in the sense orientation behindthe Kti promoter, to DNA of KS126 (PCT Publication No. WO 04/071467)linearized with the restriction enzyme NotI to give KS308 (SEQ IDNO:41).

The 3.1 kb DNA fragment containing β-conglycinin promoter, HGGT gene,and phaseolin terminator was excised from SC38 (see Example 3) using therestriction enzyme AscI, the ends were blunted with the large fragmentof DNA polymerase I, and ligated to DNA of KS178 (construction describedbelow) to give KS270 (SEQ ID NO:42). KS178 had previously beenlinearized with the restriction enzyme Pad followed by filling in of 3′overhangs with the large fragment of DNA polymerase I.

KS178 was constructed as follows. The 4.0 kb DNA fragment containing theSAMS/ALS/ALS3′ cassette, was excised from pZSL13LeuB (PCT PublicationNo. WO 04/071467) using the restriction enzymes PstI and SmaI, the endswere blunted with the large fragment of DNA polymerase I, and ligated toDNA of KS102 (PCT Publication No. WO 04/071467) linearized with therestriction enzyme BamHI, to give KS178. Prior to ligation the ends ofthe linearized KS102 vector were blunted with the large fragment of DNApolymerase I.

The 3.4 kb DNA fragment containing the gamma-tocopherolmethyltransferase expression cassette was excised from KS308 using therestriction enzyme AscI, the ends were blunted with the large fragmentof DNA polymerase I, and ligated to DNA of pBLUESCRIPT® II KS−(Stratagene) linearized with the restriction enzyme SmaI. The resultingvector was linearized with the restriction enzyme SnaBI, and ligated tothe 3.0 kb DNA fragment containing the HGGT expression cassette removedfrom KS270 using the restriction enzymes Pad and BamHI to give KS318.Prior to ligation the ends of this fragment were blunted with the largefragment of DNA polymerase I. The 6.4 kb DNA fragment containing theHGGT and gamma-tocopherol methyltransferase expression cassettes wasexcised from KS318 using the restriction enzyme SalI, and ligated to DNAof the Agrobacterium tumefaciens binary vector pZBL120, linearized withSalI, to give KS319. The T-DNA of the plant transformation vector KS319is set forth as SEQ ID NO:43.

Applicants note that the binary vector pZBL120 is identical to the pZBL1binary vector (American Type Culture Collection Accession No. 209128)described in U.S. Pat. No. 5,968,793, except the NOS promoter wasreplaced with a 963 bp 35S promoter (NCBI Accession No. V00141; alsoknown as NCBI General Indentifier No. 58821) from nucleotide 6494 to7456 in the Nos/P-nptII-OCS 3′ gene. The new 35S promoter-nptII-OCS 3′gene serves as a kanamycin (Kan) resistance plant selection marker inpZBL120.

Generation and Analysis of Transgenic Arabidospis Lines:

Plasmid DNA of KS319 was introduced into Agrobacterium tumefaciens NTL4(Luo et al, Molecular Plant-Microbe Interactions (2001) 14(1):98-103) byelectroporation. Briefly, 1 μg plasmid DNA was mixed with 100 μL ofelectro-competent cells on ice. The cell suspension was transferred to a100 μL electro oration curette (1 mm gap width) and electro orated usinga BIORAD electro orator set to 1 kV, 400Ω and 25 μF. Cells weretransferred to 1 mL LB medium and incubated for 2 h at 30° C. Cells wereplated onto LB medium containing 50 μg/mL kanamycin. Plates wereincubated at 30° C. for 60 h. Recombinant agrobacterium cultures (500 mLLB, 50 μg/mL kanamycin) were inoculated from single colonies oftransformed agrobacterium cells and grown at 30° C. for 60 h. Cells wereharvested by centrifugation (5000×g, 10 min) and resuspended in 1 L of5% (W/V) sucrose containing 0.05% (V/V) Silwet. Arabidopsis plants weregrown in soil at a density of 30 plants per 100 cm² pot in METRO-MIX®360 soil mixture for 4 weeks (22° C., 16 h light/8 h dark, 100 μEm⁻²s⁻¹). Plants were repeatedly dipped into the agrobacterium suspensionharboring the binary vector KS319 and kept in a dark, high humidityenvironment for 24 h. Plants were grown for three to four weeks understandard plant growth conditions described above and plant material washarvested and dried for one week at ambient temperatures in paper bags.Seeds were harvested using a 0.425 mm mesh brass sieve.

Cleaned Arabidopsis seeds (2 grams, corresponding to about 100,000seeds) were sterilized by washes in 45 mL of 80% ethanol, 0.01% tritonX-100, followed by 45 mL of 30% (V/V) household bleach in water, 0.01%triton X-100 and finally by repeated rinsing in sterile water. Aliquotsof 20,000 seeds were transferred to square plates (20×20 cm) containing150 mL of sterile plant growth medium comprised of 0.5×MS salts, 1.0%(W/V) sucrose, 0.05 MES/KOH (pH 5.8), 200 μg/mL timentin, and 50 μg/mLkanamycin solidified with 10 g/L agar. Homogeneous dispersion of theseed on the medium was facilitated by mixing the aqueous seed suspensionwith an equal volume of melted plant growth medium. Plates wereincubated under standard growth conditions for ten days.Kanamycin-resistant seedlings were transferred to plant growth mediumwithout selective agent and grown to maturity.

A total of 137 transgenic lines were generated and subjected to HPLCanalysis: 5 mg crushed seed were extracted at ambient temperature in 200μL of heptane. Tocopherols and tocotrienols were quantitated by HPLC asdescribed in Example 3. The highest total tocotrienol content was 2,800ppm. The highest alpha-tocotrienol content was 400 ppm. In these events,25% of all tocopherols and tocotrienols comprised of alpha-tocotrienol.

Two events, #58 and #135 were advanced to transgene homozygousity byrepeated selfing. T2 seed of both events contained 25% ofkanamycin-sensitive seed indicating that both events contained transgeneinsertion at a single genetic locus. Bulk seed were produced from T3seed that no longer segregated kanamycin-sensitive progeny. Fifty mg ofT4 seed material was extracted in 1 mL of heptane. Tocopherol andtocotrienol were quantitated by HPLC and these results are found inTable 5. As discussed below, event #58 expressed HGGT andgamma-tocopherol methyltransferase genes. Event #135 expressed onlyHGGT.

TABLE 5 Tocol Composition (% of total tocols) of Homozygous T4 SeedMaterial of Transgenic Arabidopsis Lines alpha- beta- gamma- tocoph-tocoph- tocoph- delta- line erol erol erol tocopherol Tocopherolwild-type ppm 6 0 396 9 411 % 2 0 96 2 #135 ppm 25 4 326 104 455 % 1 012 4 #58 ppm 308 58 98 30 495 % 15 3 5 1 wild-type ppm 0 0 0 0 0 % 0 0 00 #135 ppm 13 0 1754 590 2358 % 0 0 62 21 #58 ppm 419 47 876 273 1615 %20 2 42 13

Table 5 indicates that event #135 apparently only expresses the barleyHGGT gene. The seed tocotrienol profile of event #135 resembles that ofleaves of transgenic Arabidopsis plants over-expressing the barley HGGTgene. The leaf profile is dominated by gamma-tocotrienol withalpha-tocotrienol comprising less than 3% of the total tocotrienolfraction (see PCT Publication No. WO 03/082899; U.S. Application No.2004/0034886; Cahoon et al. (2003) Nat. Biotechnol. 21:1082-1087).Applicants note that in line #135 only trace levels of alpha-tocotrienolare detected. Hence, there is very little endogenous enzyme activitypresent in Arabidopsis seed that can convert gamma-tocotrienol toalpha-tocotrienol.

In contrast to the above, the co-expression of the soybeangamma-tocopherol methyltransferase gene with the HGGT gene in event #58leads to significant accumulation of alpha-tocotrienol with levels of419 ppm. The oil content of heptane extracts was measured using sodiummethoxide derivatization followed by GC analysis (see below). Using thisanalysis, it was determined that the seed oil of event #58 contained1,200 ppm alpha-tocotrienol. The alpha-tocotrienol of event #58 makes upabout 20% of the total tocopherols and tocotrienols. About 30% ofgamma-tocotrienol is converted to alpha-tocotrienol. Applicants notethat expression of the gamma-tocopherol methyltransferase gene may below, because a heterologous promoter was used. Even higher levels ofalpha-tocotrienol will very likely be observed if the gamma-tocopherolmethyltransferase gene is expressed under control of an endogenousseed-preferred promoter. Nevertheless, the Arabidopsis data hasdemonstrated that the soybean gamma-tocopherol methyltransferase gene isan efficient enzyme catalyst for methylation of tocotrienols for theproduction of alpha- and beta-tocotrienol.

One skilled in the art understands that the homogentisate geranylgeranyltransferases (HGGT) and gamma-tocopherol methyltransferases found inTable 1 and Table 2, respectively, may also be expressed in Arabidopsisthaliana to demonstrate the feasability of using these cDNA to increasealpha and beta-tocotrienol production in transgenic plants.

GC/MS Analysis to Confirm Identity of Tocopherols and Tocotrienols:

Total tocol analysis was performed on an Agilent 6890 gas chromatographin conjunction with Agilent 5973 Mass Selective Detector (MSD). Four μLsamples of heptane extracts of Arabidopsis seeds of lines #58 and #135were injected into a split/splitless injector (2:1 split ratio) held at300° C. Chromatographic separation was performed on a 30 m×250 μm(ID)×0.25 μm (film thickness) Agilent DB5MS column using helium gas asthe carrier (39 cm/sec linear velocity). The oven temperature profilewas as follows: 260° C., hold 4 min; 2° C. ramp to 340° C., hold for 12min. Compounds eluting from the column were directed into the MSD thougha heated (325° C.) transfer line and ionized (70 eV). The MSD was tunedusing the standard tune protocol and was run in Scan mode (10-500 massrange). Data was analyzed using ChemStation (Agilent) and AMDIS version2.1 (National Institute of Standards and Technology; NIST).

Compound identity was confirmed by comparing compound elution times withthose of authentic samples and by mass spectral comparisons with anelectronic database (version 2.0, NIST). The database contained entriesfor alpha-, beta-, gamma- and delta-tocopherols, as well as the internalstandard (alpha-tocopherol acetate). Library entries were not availablefor any of the tocotrienols. The identity of these compounds wastherefore confirmed by comparison of the chromatographic elution and byvisual comparison of the mass-spectrum with those of authentic standardsrun under the same chromatographic conditions.

Example 2 Production of Tocotrienols in Transgenic Soybean LinesMolecular Stack of Barley HGGT and Soybean Gamma-TocopherolMethyltransferase

To demonstrate the ability to produce increased levels ofalpha-tocotrienols, beta-tocotrienols, or both, in transgenic soybeanlines, the barley HGGT cDNA (bdl2c.pk006.o2; SEQ ID NO:2) and soybeangamma-tocopherol methyltransferase (sah1c.pk004.g2; SEQ ID NO:12) wereused in a molecular stack (progeny with both transgene-related traits).

Transgenic soybean lines were generated with plasmid DNA of KS270 andKS308, see Example 1, using particle bombardment of embryogenic callus.

KS270 provides the barley HGGT gene under control of 617 bp of thesoybean O-conglycinin promoter. The polyadenylation signal for the HGGTtranscript is derived from the terminator of the phaseolin gene (fromthe bean Phaseolus vulgaris; Doyle et al. (1986) J. Biol. Chem.261:9228-9238). The plasmid also contains the cDNA of asulfonylurea-resistant variant of the soybean ALS gene that is undercontrol of 1217 bp of the SAMS promoter. The polyadenylation signal forthe HGGT transcript is derived from the terminator of the soybean ALSgene.

KS308 provides the gamma-tocopherol methyltransferase gene from soybeanunder the control of 2090 bp of the soybean Kti promoter. Thepolyadeylation signal for the gamma-tocopherol methyltransferasetranscript is derived from the terminator of the Kti gene. KS308 alsoprovides a hygromycin B phosphotransferase (HPT) resistance gene (Gritzet al. (1983) Gene 25:179-188) that is under control of 1408 bp of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812). The polyadenylation signal for the hygromycin resistancegene is derived from the terminator of nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

Soybean embryogenic suspension cultures were transformed with DNAplasmids KS270 in conjunction with KS308 by the method of particle gunbombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat.No. 4,945,050) using a BIORAD BIOLISTIC™ PDS1000/He instrument. Thefollowing stock solutions and media were used for transformation andregeneration of soybean plants:

Stock Solutions:

-   Sulfate 100× Stock: 37.0 g MgSO₄.7H₂O, 1.69 g MnSO₄.H₂O, 0.86 g    ZnSO₄.7H₂O, 0.0025 g CuSO₄.5H₂O-   Halides 100× Stock: 30.0 g CaCl₂.2H₂O, 0.083 g KI, 0.0025 g    CoCl₂.6H₂O-   P, B, Mo 100× Stock: 18.5 g KH₂PO₄, 0.62 g H₃BO₃, 0.025 g    Na₂MoO₄.2H₂O-   Fe EDTA 100× Stock: 3.724 g Na₂EDTA, 2.784 g FeSO₄.7H₂O-   2,4-D Stock: 10 mg/mL-   Vitamin B5 1000× Stock: 10.0 g myo-inositol, 0.10 g nicotinic acid,    0.10 g pyridoxine HCl, 1 g thiamine.    Media (per Liter):-   SB196: 10 mL of each of the above stock solutions, 1 mL B5 Vitamin    stock, 0.463 g (NH₄)₂SO₄, 2.83 g KNO₃, 1 mL 2,4-D stock, 1 g    asparagine, 10 g Sucrose, pH 5.7-   SB103: 1 pk. Murashige & Skoog salts mixture, 1 mL B5 Vitamin stock,    750 mg MgCl₂ hexahydrate, 60 g maltose, 2 g GELRITE®, pH 5.7.-   SB166: SB103 supplemented with 5 g per liter activated charcoal.-   SB71-4: Gamborg's B5 salts, 1 mL B5 vitamin stock, 30 g sucrose, 5 g    TC agar, pH 5.7.

To prepare tissue for transformation, soybean embryogenic suspensioncultures were maintained in 35 mL liquid medium (SB196) on a rotaryshaker (150 rpm) at 28° C. with fluorescent lights providing a 16 hourday/8 hour night cycle. Cultures were subcultured every 2 weeks byinoculating approximately 35 mg of tissue into 35 mL of fresh liquidmedia.

In particle gun bombardment procedures it is possible to use purified 1)entire plasmid DNA or, 2) DNA fragments containing only the recombinantDNA expression cassette(s) of interest. For every seventeen bombardmenttransformations, 85 μL of suspension is prepared containing 1 to 90picograms (pg) of plasmid DNA per base pair of each DNA plasmid. Bothrecombinant DNA plasmids were co-precipitated onto gold particles asfollows. The DNAs in suspension were added to 50 μL of a 20-60 mg/mL 0.6μm gold particle suspension and then combined with 50 μL CaCl₂ (2.5 M)and 20 μL spermidine (0.1 M). The mixture was vortexed for 5 seconds,spun in a microfuge for 5 seconds, and the supernatant removed. TheDNA-coated particles were then washed once with 150 μL of 100% ethanol,vortexed and spun in a microfuge again, then resuspended in 85 μL ofanhydrous ethanol. Five μL of the DNA-coated gold particles were thenloaded on each macrocarrier disk.

Approximately 150 to 250 mg of two-week-old suspension culture wasplaced in an empty 60 mm×15 mm petri plate and the residual liquidremoved from the tissue using a pipette. The tissue was placed about 3.5inches away from the retaining screen and each plate of tissue wasbombarded once. Membrane rupture pressure was set at 650 psi and thechamber was evacuated to −28 inches of Hg. Three plates were bombarded,and, following bombardment, the tissue from each plate was dividedbetween two flasks, placed back into liquid media, and cultured asdescribed above.

Seven days after bombardment, the liquid medium was exchanged with freshSB196 medium supplemented with 30-50 mg/L hygromycin. The selectivemedium was subsequently refreshed weekly or biweekly. Seven weekspost-bombardment, bright green, transformed tissue was observed growingfrom untransformed, chlorotic or necrotic embryogenic clusters. Isolatedgreen tissue was removed and inoculated into individual wells insix-well culture dishes to generate new, clonally-propagated,transformed embryogenic suspension cultures. Thus, each new line wastreated as independent transformation event in an individual well. Thesesuspensions can then be maintained as suspensions of embryos clusteredin an immature developmental stage through subculture or they can beregenerated into whole plants by maturation and germination ofindividual somatic embryos.

After two weeks in individual cell wells, transformed embryogenicclusters were removed from liquid culture and placed on solidifiedmedium (SB166) containing no hormones or antibiotics for one week.Embryos were cultured for at 26° C. with mixed fluorescent andincandescent lights on a 16 hour day/8 hour night schedule. After oneweek, the cultures were then transferred to SB103 medium and maintainedin the same growth conditions for 3 additional weeks.

Somatic embryos became suitable for germination after 4 weeks and werethen removed from the maturation medium and dried in empty petri dishesfor 1 to 5 days. The dried embryos were then planted in SB71-4 mediumwhere they were allowed to germinate under the same light andtemperature conditions as described above. Germinated embryos weretransferred to sterile soil and grown to maturity for seed production.

A total of fourteen events were created by co-transformation with KS270and KS308 plasmids. Tocol composition of T1 seed was assayed as follows.A seed chip (approximately 5-15 mg of tissue) was obtained from thecotyledon tissue of the seed. The chip was extracted with 100 μL ofheptane for 2 hours. Tocopherol and tocotrienol was quantitated by HPLCanalysis as described in Example 3.

A total of 14 events were generated and analyzed. Seed from five eventscontained significant levels of tocotrienol. Three of these alsocontained significant levels (>150 ppm) of alpha- and beta-tocotrienol.One event did not show conversion of gamma- to alpha-tocotrienol and oneevent did only exhibit low levels of gamma-tocopherol methyltransferaseactivity (20-150 ppm alpha-tocotrienol). One event 4060.2.5.1 wasselected for further work. For event 4060.2.5.1, seven out of ten T1seed showed the transgenic trait, indicating that these events likelyhad a single or multiple transgenic insertion at a single genetic locus.Positive-positive T1 seed were planted and T2 seed were selected fromindividual plants. A total of forty-eight T2 seed was analyzed by HPLCand the results can be found in Table 6.

TABLE 6 Tocol Composition (% of total tocopherols (tocph.) andtocotrienols (toct.)) for T2 Progeny of Event 4060.2.5.1 alpha- beta-gamma- delta- alpha- beta- gamma- delta- tocph. toct. No. tocph. tocph.tocph. tocph. toct. toct. toct. toct. (ppm) (ppm) 1 8 4 0 0 31 24 12 20406 2786 2 9 5 0 0 31 22 15 19 474 3100 3 8 4 0 0 30 24 13 21 453 3172 49 4 0 0 30 23 14 20 471 2922 5 7 4 0 0 30 24 13 21 389 3059 6 9 5 0 0 2922 13 22 479 3046 7 9 5 0 0 29 23 12 22 434 2596 8 9 5 0 0 29 22 13 22454 2693 9 9 5 0 0 28 22 13 22 442 2595 10 10 5 0 0 28 22 13 22 487 268611 8 5 0 0 28 22 15 21 292 1846 12 10 5 0 0 27 22 12 23 401 2120 13 10 50 0 27 23 12 23 384 2164 14 10 5 0 0 27 19 17 23 424 2481 15 8 3 0 0 2614 26 22 382 2912 16 8 5 0 0 26 22 14 26 468 3128 17 8 5 0 0 26 22 14 26399 2692 18 9 5 0 0 25 21 14 25 477 2906 19 7 5 0 0 25 23 13 26 365 258020 7 5 0 0 25 21 14 27 405 2826 21 7 5 0 0 25 22 14 27 442 3138 22 11 50 0 24 16 19 24 408 2084 23 8 6 0 0 24 22 14 27 435 2818 24 7 5 0 0 2420 15 29 411 2947 25 9 6 0 0 24 21 13 27 412 2340 26 9 6 0 0 24 20 15 27453 2624 27 9 6 0 0 23 21 14 27 392 2315 28 9 6 0 0 23 20 14 28 443 241529 8 2 1 0 22 10 36 21 460 3873 30 7 5 0 0 22 21 14 30 386 2723 31 9 5 00 22 18 17 30 435 2718 32 16 1 73 10 0 0 0 0 383 0 33 51 2 45 2 0 0 0 0368 0 34 35 2 59 4 0 0 0 0 362 0 35 20 1 69 10 0 0 0 0 353 0 36 36 2 565 0 0 0 0 325 0 37 18 2 71 10 0 0 0 0 357 0 38 35 3 58 5 0 0 0 0 307 039 13 2 74 11 0 0 0 0 302 0 40 25 2 64 9 0 0 0 0 353 0 41 18 1 71 10 0 00 0 328 0 42 25 2 64 9 0 0 0 0 353 0 43 17 2 70 11 0 0 0 0 384 0 44 14 173 12 0 0 0 0 337 0 45 20 1 70 8 0 0 0 0 344 0 46 16 1 73 10 0 0 0 0 3350 47 16 1 74 10 0 0 0 0 328 0 48 18 1 71 8 0 0 0 2 354 0

The T2 seed were generated through selfing of a transgenic line that washeterzogous for a single dominant transgenic trait. Accordingly, onewould expect to detect 25% (12/48) non-transgenic segregants. Applicantsobserved 35% (17/48) non-transgenic segregants (see numbers 32-48).Seeds numbers 1 to 31 are transgenic segregants.

T2 progeny with both transgene-related traits were found to contain atleast 590 ppm and as much as 1,099 ppm alpha-tocotrienol and at least401 ppm and as much as 868 ppm beta-tocotrienol. In these T2 lines,alpha-tocotrienol constituted at least 22% and up to 31%, and integersin between, of the total tocopherol and tocotrienol fraction. Oilcontent of the heptane extracts was determined by derivatization withsodium methoxide followed by GC analysis. Oil could be calculated fromthat tocotrienol concentrations expressed as ppm. T2 progeny with bothtransgene-related traits contained an oil with at least 2,618 ppm and asmuch as 4,891 alpha-tocotrienol and at least 1,732 ppm and as much as3,804 ppm beta-tocotrienol. Applicants also tested for a possiblenegative effect of the high alpha- and beta-tocotrienol content on seedweight. To this end, seed weight of the forty-eight T2 seed was plottedagainst alpha-tocotrienol content. No correlation between seed weightand alpha-tocotrienol content could be detected. Moreover, no unusualseed phenotypes related to seed shape, coloration or germinationbehaviour were observed in seed with the high alpha- andbeta-tocotrienol trait.

One skilled in the art understands that the homogentisate geranylgeranyltransferases (HGGT) and gamma-tocopherol methyltransferases found inTable 1 and Table 2, respectively, may also be expressed in soybean todemonstrate the feasability of these cDNA for alpha- andbeta-tocotrienol production in transgenic plants.

In summary, gamma-tocopherol methyltransferase enzyme from soybean canefficiently use tocotrienol substrates, for example, by the foregoingmethod to generate a seed or an extracted oil with high levels of alpha-and beta-tocotrienol. The alpha-tocotrienol content of soybeansover-expressing barley HGGT and the soybean gamma-tocopherolmethyltransferase gene exceeds that of any non-transgenic seed or oildescribed previously by at least one order of magnitude (Packer et al.(2001) J. Nutr. 131:369 S-373S; Bertoli et al. (1998) JAOCS75:1037-1040; PCT Publication No. WO 00/072862). These results furtherdemonstrate the ability to produce alpha- and beta-tocotrienols in acrop plant that does not normally accumulate these antioxidant moleculesthrough the transgenic expression of nucleic acid fragments encodingHGGT and gamma-tocopherol methyltransferase polypeptides.

Example 3 Production of Tocotrienols in Somatic Soybean Embryos andTransgenic Soybean Lines Genetic Crossing of Barly HGGT and SoybeanGamma-Tocopherol Methyltransferase

To demonstrate the ability to produce increased levels of alpha- andbeta-tocotrienols in somatic soybean embryos and transgenic soybeanlines, the barley HGGT cDNA (bdl2c.pk006.o2; SEQ ID NO:2) and soybeangamma-tocopherol methyltransferase (sah1c.pk004.g2; SEQ ID NO:12) wereused in a genetic stack (progeny with both transgene-related traitsproduced by crossing).

Somatic soybean embryos have been used as model for the prediction oftransgenic phenotypes in soybean seeds (Kinney, A. J. (1996) J. FoodLipids 3:273-292). Somatic soybean embryos and seeds are enriched intocopherols, but contain little or no tocotrienols (Coughlan,unpublished result; The Lipid Handbook, 2nd Edition, Gunstone, F. D., etal., Eds., Chapman and Hall, London, 1994, pp. 129-131).

Plasmid DNA from clone sah1c.pk004.g2 was used as a template to preparea NotI PCR fragment encoding the entire deduced open reading frame usingthe following PCR primers: forward primer5′-AGCGCGGCCGCATGGCCACCGTGGTGAGGATCCCA-3′ (SEQ ID NO:44), AND reverseprimer 5′-AGCGCGGCCGCTTATTCAGGTTTTCGACATGTAATGATG-3′ (SEQ ID NO:45).

PCR amplification was achieved using Pfu polymerase, and DNA of ESTsah1c.pk004.g2 was used as the template. The product of this PCRreaction was purified by agarose gel electrophoresis and subcloned intopCR-Script-AMP (Stratagene) as described in the manufacturer's protocol.The amplified open-reading frame of the soybean gamma-tocopherolmethyltransferase gene was then released as a NotI fragment and clonedinto the corresponding site of soybean expression vector pKS67 togenerate plasmid pSC1 (SEQ ID NO:50). The plasmid pKS67 was prepared byreplacing in pRB20 (described in U.S. Pat. No. 5,846,784, incorporatedherein by reference) the 800 bp Nos 3′ fragment, with the 285 bp Nos 3′fragment containing the polyadenylation signal sequence and described inDepicker et al. (1982) J. Mol. Appl. Genet. 1:561-573. Ligation productswere transformed into E. coli and recombinant clones were selected usinghygromycin B selection.

Restriction digestion of plasmid DNA was used to identify culturesharboring plasmid DNA in which the start codon of the soybeangamma-tocopherol methyltransferase cDNA was in close proximity to thetranscription start site of the soybean β-conglycinin promoter. In thisplasmid construct, henceforth referred to as SC1, the soybeangamma-tocopherol methyltransferase cDNA is under the control of a 617 bpfragment of the soybean β-conglycinin promoter. The polyadenylationsignal for the HGGT transcript is derived from the terminator of thephaseolin gene. Plasmid SC1 (SEQ ID NO:50) contains hygromycin Bphosphotransferase gene under control of the cauliflower mosaic 35Spromoter, which allows for selection of transformed plant cells byresistance to the antibiotic hygromycin B. Plasmid DNA of SC1 was usedto generate transgenic somatic embryos of soybean as described below.

Transformation of Soybean Somatic Embryo Cultures:

The following stock solutions and media were used for transformation andpropagation of soybean somatic embryos:

TABLE 7 Stock Solutions and Media for Transformation and Propagation ofSoybean Somatic Embryos Stock Solutions (g/L) MS Sulfate 100x stockMgSO₄•7H₂O 37.0 MnSO₄•H₂O 1.69 ZnSO₄•7H₂O 0.86 CuSO₄•5H₂O 0.0025 MSHalides 100x stock CaCl₂•2H₂O 44.0 KI 0.083 CoCl₂•6H₂O 0.00125 KH₂PO₄17.0 H₃BO₃ 0.62 Na₂MoO₄•2H₂O 0.025 Na₂EDTA 3.724 FeSO₄•7H₂O 2.784 B5Vitamin stock myo-inositol 100.0 nicotinic acid 1.0 pyridoxine HCl 1.0thiamine 10.0 Media SB55 (per Liter) 10 mL of each MS stock 1 mL of B5Vitamin stock 0.8 g NH₄NO₃ 3.033 g KNO₃ 1 mL 2,4-D (10 mg/mL stock)0.667 g asparagine pH 5.7 SB 103 (per Liter) 1 pk. Murashige & Skoogsalt mixture* 60 g maltose 2 g GELRITE ® pH 5.7 SB148 (per Liter) 1 pk.Murashige & Skoog salt mixture* 60 g maltose 1 mL B5 vitamin stock 7 gagarose pH 5.7 *(Gibco BRL)

Soybean embryonic suspension cultures were maintained in 35 mL liquidmedia (SB55) on a rotary shaker (150 rpm) at 28° C. with a mix offluorescent and incandescent lights providing a 16 hour day/8 hour nightcycle. Cultures were subcultured every 2 to 3 weeks by inoculatingapproximately 35 mg of tissue into 35 mL of fresh liquid media.

Soybean embryonic suspension cultures were transformed with the plasmidcontaining the gamma-tocopherol methyltransferase sequence by the methodof particle gun bombardment (see Klein et al. (1987) Nature 327:70-73)using a DUPONT™ BIOLISTIC™ PDS1000/He instrument. Five μL of pKS93splastid DNA (1 mg/L), 50 μL Cal₂ (2.5 M), and 20 μL spermdine (0.1 M)were added to 50 μL of a 60 mg/mol 1 mm gold particle suspension. Theparticle preparation was agitated for 3 minutes, spun on a microphagefor 10 seconds and the supernatant removed. The DNA-coated particleswere then washed once with 400 μL of 70% ethanol and resuspended in 40μL of anhydrous ethanol. The DNA/particle suspension was sonicated threetimes for 1 second each. Five μL of the DNA-coated gold particles werethen loaded on each macro carrier disk.

Approximately 300 to 400 mg of two-week-old suspension culture wasplaced in an empty 60 mm×15 mm petri dish and the residual liquidremoved from the tissue using a pipette. The tissue was placed about 3.5inches away from the retaining screen and bombarded twice. Membranerupture pressure was set at 1100 psi and the chamber was evacuated to−28 inches of Hg. Two plates were bombarded, and following bombardment,the tissue was divided in half, placed back into liquid media, andcultured as described above.

Fifteen days after bombardment, the liquid media was exchanged withfresh SB55 containing 50 mg/mL hygromycin. The selective media wasrefreshed weekly. Six weeks after bombardment, green, transformed tissuewas isolated and inoculated into flasks to generate new transformedembryonic suspension cultures.

Transformed embryonic clusters were removed from liquid culture mediaand placed on a solid agar media, SB103, containing 0.5% charcoal tobegin maturation. After one week, embryos were transferred to SB103media minus charcoal. After five weeks on SB103 media, maturing embryoswere separated and placed onto SB148 media. During maturation embryoswere kept at 26° C. with a mix of fluorescent and incandescent lightsproviding a 16 hour day/8 hour night cycle. After three weeks on SB148media, embryos were analyzed for the expression of the tocopherols. Eachembryonic cluster gave rise to 5 to 20 somatic embryos.

Non-transformed somatic embryos were cultured by the same method as usedfor the transformed somatic embryos.

Analysis of Transformed Somatic Embryos:

At the end of the sixth week on SB148 medium, somatic embryos wereharvested from 25 independently transformed lines. Somatic embryos werecollected in pools of five and weighed for fresh weight. Excess embryoswere stored in 96-well plates at −80° C. The pooled somatic embryos werelyophilized for 18 hours and the dry weight measured. The lyophilizedsomatic embryos were briefly pulverized with a hand held Potterhomogeniser and then 600 μL of heptane added and the samples incubatedfor 24 hours in the dark at room temperature to extract oils andtocopherols. The heptane was decanted and a further 300 μL added to thesamples. The extracts were combined and centrifuged (5 minutes, 12000g). The supernatant was stored in amber hulk auto sampler vials at −20°C. prior to analysis.

HPLC analysis of the extracts was carried out using an HP1100 system(Agilent Technologies) 25 μL of the heptane sample was applied to aLichrosphere Si 60 column (5 micron, 4×12.5 mm). The column was elutedwith heptane/isopropanol (98:2 v/v) at a flow rate of 1 mL/min. Aftersix minutes all four tocopherol isomers were eluted, as detected by aHP1100 fluorescence detector (Excitation wavelength 295 nm, emissionwavelength 330 nm). Individual tocopherol standards (Matreya) werediluted with HPLC grade heptane to levels between 1 and 200 ng/μL toconstruct a 6-point external standard curve. Tocopherols in each oilwere quantified using a standard curve run on the same day as thesamples. The sum of tocopherol peak areas of samples from anon-transformed control line were compared with those of 25 independentgamma-tocopherol methyltransferase-transformed, hygromycin resistantlines.

Several events were identified that showed over-expression of thesoybean gamma-tocopherol methyltransferase gene. In many of the lines80% of the total tocol fraction was comprised of alpha-tocopherol incontrast to untransformed soybean embryos where gamma-tocopherolconstitutes the dominant tocol molecule. Soybean plants were generatedfrom clonal tissue derived from ten independent transgenic soybeanevents with high levels of alpha-tocopherol. Several plants weregenerated for each of the ten events. Five T1 seed from each transgenicevent were subjected to HPLC analysis to determine the composition ofthe tocopherol fraction. Briefly, individual dry beans were homogenizedusing a tissue pulverizer (Genogrinder). Approximately 30 mg of tissuepowder were extracted with 600 μL for 2 hours at ambient temperature.The heptane extract was cleared by brief centrifugation. Tocolcomposition of the heptane extracts was analyzed by HPLC as describedpreviously. Percent alpha-tocopherol of T1 seed is summarized in Table8.

TABLE 8 Percent Alpha-Tocopherol of T1 Seed Seed # Seed # Seed # Seed #Seed # Event 1 2 3 4 5 719/1/1/A 5.8 4.9 4.2 6.9 6.7 719/1/1/B 8.4 5.67.0 8.8 7.6 719/1/1/C 6.3 2.8 2.5 4.8 5.7 719/1/2/A 52.4 56.5 53.0 47.051.1 719/1/2/B 56.7 5.0 43.9 11.4 5.4 719/1/2/C 41.9 44.4 2.9 42.7 5.9719/1/3/A 18.4 14.8 22.5 6.5 16.7 719/1/4/A 7.0 5.3 11.7 5.6 10.6719/1/4/B 2.5 4.9 2.0 5.3 1.0 719/1/5/A 34.1 52.9 31.2 37.6 9.2719/1/5/B 7.7 10.4 61.7 60.9 57.8 719/1/8/A 30.7 15.2 33.9 42.4 53.1719/1/10/A 8.2 75.0 86.0 79.4 80.5 719/1/10/B 85.3 81.2 8.0 7.4 80.1719/1/10/C 80.4 79.0 80.0 83.8 86.8 719/1/13/A 14.6 9.1 7.3 10.2 9.2719/1/13/B 4.5 83.0 6.0 81.3 7.2 719/1/13/C 78.1 9.5 9.7 9.2 10.7719/1/13/D 12.8 11.4 11.5 7.6 10.8 719/1/13/E 8.5 11.5 14.2 14.0 10.9720/4/1/A 16.4 6.1 7.1 5.1 8.9 720/4/2/A 7.2 79.6 73.1 50.9 34.7720/4/2/B 58.3 54.6 52.9 51.7 62.6 720/4/2/C 7.0 53.7 59.8 79.1 42.7721/7/1/A 8.4 6.6 7.2 6.4 8.7

Event 719.1.10 was selected for advancement. The segregation of the highalpha-tocopherol trait in T1 seed indicated that this event has a singlelocus insertion of the over-expressed gamma-tocopherol methyltransferasegene. T1 plants were allowed to self and T2 seed selections fromindividual plants were subjected to HPLC analysis of individual seed. T2seed selections were identified that no longer segregated seed with thelow alpha-tocopherol content (alpha-tocopherol <10% of total tocol).Seed from these selections were planted and bulk seed that werehomozygous of the transgene were harvested from these T2 plants.

Quantitative analysis of tocopherols of T3 seed was conducted asfollows. Soybeans were ground in a FOSS tecator sample mill (FOSS, USA)using a 1 mm screen. 200 mg of tissue were extracted in 5 mL of heptanefor two hours; alpha-tocopherol acetate was added as internal standardat a final concentration of 38 μg mL⁻¹. Ten μL of filtered heptaneextract was subjected to HPLC using a Lichrospher column (250-4 HPLCcartridge, Si60, 5 μM particle size) using heptane containing 0.75%isopropanol as mobile phase at a flow rate of 1 mL min⁻¹. Externalstandards of all four tocopherols and tocotrienols (2.5 μg mL⁻¹)separated under identical conditions were used for tocol quantitation.Tocols were detected using a fluorescence detector using excitation andemission wavelengths of 295 nm, 330 nm, respectively. Table 9 indicatesthat EMSP 719.1.10 expresses high level of gamma-tocopherolmethyltransferase activity indicated by the nearly quantitativeconversion of gamma- and delta- to alpha- and beta-tocopherol,respectively. Applicants note that no tocotrienols could be detected.

TABLE 9 Tocol Composition of Homozygous T3 Seed of Event EMSP 719.1.10alpha- beta- gamma- delta- tocopherol tocopherol tocopherol tocopheroltocopherol ppm 148 29 5 3 183 % 77 15 2 1

Generation of a Transgenic Soybean Line with Seed-preferred Expressionof the Barley HGGT Gene:

A DNA fragment was generated by PCR. The new DNA fragment contains thecomplete open reading frame (1224 bp; SEQ ID NO:46) of the barley HGGTcDNA flanked at 5′ and 3′ position by DNA sequences recognized by therestriction enzyme NotI. Briefly, the modified HGGT cDNA was amplifiedfrom a barley developing seed cDNA library (see PCT Publication No. WO03/082899) using oligonucleotide primers that include NotI sites thatstart four nucleotides upstream of the start codon and two nucleotidesdownstream of the stop codon of the HGGT cDNA sequence, respectively.The sequences of the sense and antisense oligonucleotide primers used inthis reaction were as follows:

-   5′-ttgcggccgcAGGATGCAAGCCGTCACGGCGGCAGCCG-3′ (SEQ ID NO:47) and-   5′-ttgcggccgcTTCACATCTGCTGGCCCTTGTAC-3′ (SEQ ID NO:48).    (Note: The lower case, underlined nucleotide sequences correspond to    added NotI restriction sites.) PCR amplification was achieved using    Pfu polymerase, and an aliquot of the barley developing seed cDNA    library described in PCT Publication No. WO 03/082899 was used as    the template. The product of this PCR reaction was purified by    agarose gel electrophoresis and subcloned into pCR-Script-AMP    (Stratagene) as described in the manufacturer's protocol.

The amplified open-reading frame of the barley HGGT was then released asa NotI fragment and cloned into the corresponding site of soybeanexpression vector pKS123 (construction described below) to generateplasmid pSC38 (SEQ ID NO:49).

The construction of vector pKS123 was previously described in PCTPublication No. WO 02/008269 (the contents of which are herebyincorporated by reference). Briefly, plasmid pKS123 contains thehygromycin B phosphotransferase gene (HPT) (Gritz, L. and Davies, J.(1983) Gene 25:179-188), flanked by the T7 promoter and transcriptionterminator (T7prom/hpt/T7term cassette), and a bacterial origin ofreplication (ori) for selection and replication in bacteria (e.g., E.coli). In addition, pKS123 also contains the hygromycin Bphosphotransferase gene, flanked by the 35S promoter (Odell et al.Nature (1985) 313:810-812) and NOS 3′ transcription terminator (Depickeret al. J. Mol. Appl. Genet. (1982) 1:561:570) (35S/hpt/NOS3′ cassette)for selection in plants such as soybean. pKS123 also contains a NotIrestriction site, flanked by the promoter for the α′ subunit ofβ-conglycinin (Beachy et al. EMBO J. (1985) 4:3047-3053) and the 3′transcription termination region of the phaseolin gene (Doyle, J. J. etal. J. Biol. Chem. (1986) 261:9228-9238) thus allowing for strongtissue-preferred expression in the seeds of soybean of genes cloned intothe NotI site.

Ligation products were transformed into E. coli and recombinant cloneswere selected using hygromycin B selection. Restriction digestion ofplasmid DNA was used to identify cultures harboring plasmid DNA in whichthe start codon of the HGGT cDNA was in close proximity to thetranscription start site of the soybean β-conglycinin promoter. In thisplasmid construct henceforth referred to as SC38, the barley HGGT cDNAis under the control of a 617 bp fragment of the β-conglycinin promoter.The polyadenylation signal for the HGGT transcript is derived from theterminator of the phaseolin gene. Plasmid SC38 contains hygromycin Bphosphotransferase gene under control of the cauliflower mosaic 35Spromoter, which allows for selection of transformed plant cells byresistance to the antibiotic hygromycin B. Plasmid DNA of SC38 was usedto generate transgenic somatic embryos of soybean as described above.

A total of 31 independent events were created. Analysis of tocopherolsand tocotrienols was performed by HPLC analysis as described above.Eight events could be identified that contained detectable levels oftocotrienols indicating that in these transgenic events the barley HGGTenzyme was expressed. Tocotrienol levels are below detection limits offluorescence detection in unmodified leaf and seed tissue of soybean.Transgenic soybeans plants were generated from somatic embryo tissue ofone event (1052.5.2). A total of eight T1 seed were subjected analysisof tocopherols and tocotrienols by HPLC of these six seed containeddetectable levels of tocotrienols. The segregation of the tocotrienoltrait in T1 seed indicated that this event contains a single locusinsertion of the β-conglycinin::HGGT expression cassette.

Nineteen randomly selected T1 seed were grown and T2 seed were selectedfrom individual plants. Initially, eight seed from each T2 progeny weresubjected to HPLC analysis. This analysis allowed Applicants to identifyfive T2 progeny that did not produce seed lacking tocotrienols. Thenon-segregating nature of these progeny was further confirmed throughanalysis of another eight seed by HPLC. One of the homozygous T2 seedselections was used to produce bulked T3 seed. This seed material wasused for quantitative tocol analysis and these results are found inTable 10. Table 10 shows that soybeans over-expressing the HGGT genefrom barley accumulate only gamma- and delta-tocotrienol. No alpha- orbeta-tocotrienol could be detected in these transgenic lines.

TABLE 10 Tocol Composition of Homozygous T3 Seed of Event EMSP 1052.5.2alpha- beta- gamma- delta- tocopherol tocopherol tocopherol tocopheroltocopherol ppm 12 7 94 82 196 % 0 0 3 3 alpha- beta- gamma- delta-tocotrienol tocotrienol tocotrienol tocotrienol tocotrienol ppm 0 0 13291212 2540 % 0 0 49 44

The tocotrienol profile of soybeans expressing the HGGT protein frombarley indicate that there is no detectable activity converting gamma-and delta-tocotrienols to alpha- and beta-tocotrienols, respectively.Although not to be limited by theory, two possible scenarios couldexplain the lack of conversion of gamma- and delta-tocotrienol to alpha-and beta-tocotrienols in HGGT-expressing seed of dicotyledoneous plantssuch as soybean. First, gamma-tocopherol methyltransferase enzymes fromplants that do not synthesize tocotrienols may not accept tocotrienolsubstrates. According to this scenario, gamma-tocopherolmethyltransferase enzymes from monocotyledoneous plants have evolvedinto catalysts for tocotrienol methylation and their co-expression withHGGT would be required for biosynthesis of high levels of alpha- andbeta-tocotrienols in dicots. Second, gamma-tocopherol methyltransferaseenzymes from dicots may be effective enzymes for synthesis of alpha- andbeta-tocotrienols, but their endogenous expression level is too low toachieve conversion of tocotrienol substrates (i.e., the gamma-tocopherolmethyltransferase enzymes may be saturated with tocopherol substratesfrom the over-expression of HGGT).

Combination of Traits for Over-expression of HGGT and Gamma-TocopherolMethyltransferase by Genetic Crossing:

EMSP 719.1.10 was crossed to EMSP 1052.5.2 to test the feasability ofthe soybean gamma-tocopherol methyltransferase enzyme for biosynthesisof alpha- and beta-tocotrienol. A total of 20 F1 seed was generated.Quantitative analysis of tocol composition of F1 seed was conducted on atotal of four F1 seed and the results are found in Table 11.

TABLE 11 Tocol Composition of F1 Seed Containing Transgenes forSeed-Preferred Over-Expression of the HGGT Gene from Barley and theGamma-Tocopherol Methyltransferase Gene From Soybean alpha- beta- gamma-delta- tocoph- tocoph- tocoph- tocoph- tocoph- erol erol erol erol erolEMSP 1052.5.2 ppm 11 5 93 74 184 % 0.8 0.4 6 5 EMSP1052.5.2; ppm 143 810 2 226 EMSP 719.1.10 % 14 8 0 0 EMSP 1052.5.2 ppm 0 0 581 681 1261 % 00 40 47 EMSP1052.5.2; ppm 274 289 54 146 763 EMSP 719.1.10 % 28 29 5 15

Comparison of the tocol profile of EMSP 1052.5.2 to that of F1 beans ofa cross of EMSP 1052.5.2 to EMSP 719.1.10 reveals dramatic differences.Whereas alpha-tocotrienol is not detectable in the 1052.5.2 parent, itconstitutes the second most abundant tocotrienol species in the crossedmaterial. Applicants note that gamma-tocotrienol is almost completelyconverted to alpha-tocotrienol. The soybean gamma-tocopherolmethyltransferase enzyme evidently also converts delta- tobeta-tocotrienol. The lower total tocotrienol concentration of the F1beans (763 ppm compared to 1,261 ppm in the 1052.5.2 parent) may beattributed to the heterozygous state of the HGGT transgene in the F1seed or could indicate that the two β-conglycinin promoter-driventranscripts are subject to transcriptional or post-transcriptional genesilencing due to identical promoter and/or 5′UTR sequences. F1 seed weregerminated in soil and allow to self. A total of forty-eight F2 seed wasanalyzed by HPLC and the results are found in Table 12.

TABLE 12 Tocol Composition (percent of total tocopherols (tocph.) andtocotrienols (toct.)) for F2 Progeny of a Cross of EMSP 1052.5.2 to EMSP719.1.10 alpha- beta- gamma- delta- alpha- beta- gamma- delta- tocph.toct. No. tocph. tocph. tocph. tocph. toct. toct. toct toct. (ppm) (ppm)1 10 7 0 0 31 37 5 10 217 1128 2 10 7 0 0 31 38 5 9 248 1220 3 10 6 0 030 36 9 10 196 1067 4 13 7 0 0 30 37 5 8 279 1124 5 9 6 0 0 30 33 9 13239 1366 6 8 6 0 0 30 39 6 11 229 1408 7 12 7 0 0 30 33 8 10 271 1098 811 7 0 0 30 34 9 9 258 1158 9 10 6 0 0 29 34 7 13 227 1177 10 15 7 0 029 31 8 9 265 903 11 10 7 0 0 28 29 12 14 199 1005 12 8 6 0 0 28 36 8 14227 1449 13 12 6 0 0 28 32 12 10 255 1144 14 9 7 0 0 28 35 8 13 227 119015 10 7 0 0 27 37 7 12 240 1196 16 13 8 0 0 27 31 7 14 263 996 17 11 7 00 27 34 8 13 228 1017 18 11 8 0 0 27 33 7 14 261 1095 19 10 7 0 0 27 367 12 256 1207 20 13 7 0 0 27 31 10 12 210 822 21 11 7 0 0 26 39 6 10 2501108 22 8 7 0 0 26 40 7 12 228 1260 23 8 6 0 0 26 35 8 17 230 1409 24 127 0 0 26 28 14 14 193 818 25 10 8 0 0 26 37 6 13 265 1155 26 7 7 0 0 2541 7 13 237 1472 27 8 7 0 0 24 38 7 15 224 1262 28 10 7 0 0 24 32 9 17282 1385 29 7 6 0 0 24 37 8 18 176 1171 30 9 7 0 0 21 29 13 21 219 111131 2 1 7 4 1 0 46 40 238 1554 32 1 0 4 3 0 0 45 45 232 2284 33 1 1 5 3 00 47 43 190 1740 34 1 1 6 4 0 0 44 44 231 1784 35 2 0 6 3 0 0 51 37 2251788 36 2 1 5 4 0 0 41 46 204 1499 37 86 14 0 0 0 0 0 0 244 0 38 84 16 00 0 0 0 0 253 0 39 83 17 0 0 0 0 0 0 183 0 40 82 18 0 0 0 0 0 0 216 0 4181 17 1 1 0 0 0 0 317 0 42 80 19 1 0 0 0 0 0 221 0 43 78 19 2 1 0 0 0 0226 0 44 34 3 56 7 0 0 0 0 225 0 45 26 3 59 11 0 0 0 0 337 0 46 23 2 6410 0 0 0 0 213 0 47 13 2 69 17 0 0 0 0 261 0 48 12 2 70 16 0 0 0 0 216 0

Tocol analysis of forty-eight F2 seed revealed 30 F2 seed that expressedboth transgene-related traits (see numbers 1-30), six and seven seedwith only HGGT or gamma-tocopherol methyltransferase traits, (seenumbers 31-36 and 37-43, respectively), and five wild-type seed (seenumbers 44-48). These findings are very close to the expectedsegregation of two unlinked, dominant traits in the F2 generation of across of two parents that were homozygous for one of each of thedominant traits. The expected frequency of F2s with both transgenictraits is 62.5% (30/48). The expected frequency of F2s with a singletransgenic trait or or no transgenic trait is 12.5% (6/48).

F2 progeny with both transgene-related traits were found to contain atleast 258 ppm and as much as 487 ppm alpha-tocotrienol and at least 278ppm and as much as 701 ppm beta-tocotrienol. The oil content of theheptane extracts was determined by derivatization with sodium methoxidefollowed by GC analysis. Oil was calculated from the tocotrienolconcentrations expressed as ppm. F2 progeny with both transgene-relatedtraits contained an oil with at least 1,670 ppm and as much as 2,940alpha-tocotrienol and at least 1,800 ppm and as much as 4,080 ppmbeta-tocotrienol. Applicants also tested for a possible negative effectof the high alpha- and beta-tocotrienol content on seed weight. To thisend, seed weight of the forty-eight F2 seed was plotted againstalpha-tocotrienol content. No correlation between seed weight andalpha-tocotrienol content could be detected.

In summary, gamma-tocopherol methyltransferase enzyme from soybean canefficiently use tocotrienol substrates, and the foregoing is a method togenerate a seed or an extracted oil with high levels of alpha- andbeta-tocotrienol. The alpha-tocotrienol content of soybeansover-expressing barley HGGT and the soybean gamma-tocopherolmethyltransferase gene exceeds that of any non-transgenic seed or oildescribed previously by at least one order of magnitude (Packer et al.(2001) J. Nutr. 131:369 S-373S; Bertoli et al. (1998) JAOCS75:1037-1040; PCT Publication No. WO 00/072862). These results furtherdemonstrate the ability to produce alpha- and beta-tocotrienols in acrop plant that does not normally accumulate these antioxidant moleculesthrough the transgenic expression of nucleic acid fragments encodingHGGT and gamma-tocopherol methyltransferase polypeptides.

Example 4 Production of Alpha- and Beta-Tocotrienols in Maize (Zea mays)Seed

Maize oil, which is derived primarily from the embryo of maize seeds, istypically enriched in tocopherols but contains little or no tocotrienols(The Lipid Handbook, 2nd Edition, Gunstone, F. D., et al., Eds., Chapmanand Hall, London, 1994, pp. 129-131). Embryo-preferred expression of thebarley HGGT gene in maize leads to accumulation of high levels oftococtrienols. 70-80% of the tocotrienols accumulate in the form ofgamma-tocotrienol and only 5-10% of the total tocotrienol fraction isrepresented by alpha-tocotrienol (see PCT Publication No. WO 03/082899;U.S. Application No. 2004/0034886, Cahoon et al. (2003) Nat. Biotechnol.21:1082-1087.

Based on results disclosed in Examples 1, 2 and 3 of the instantapplication, the barley HGGT cDNA (bdl2c.pk006.o2; SEQ ID NO:2) andsoybean gamma-tocopherol methyltransferase (sah1c.pk004.g2; SEQ IDNO:12) can be expressed in seed embryo of maize to increase the tocolantioxidant content of this tissue and the extracted oil to produce anovel tocol composition that is dominated by alpha- andbeta-tocotrienols. As described below, this result can be achieved bytransforming maize with an expression cassette comprising the soybeangamma-tocopherol methyltransferase open reading frame operably linked onits 5′ end to an embryo preferred promoter, such as the promoter for themaize 16 kDa oleosin gene (Lee, K. and Huang, A.H. (1994) Plant Mol.Biol. 26:1981-1987) and the barley HGGT open reading frame operablylinked to the maize embryo abundant (EAP1) promoter and terminator.

An expression cassette comprising the promoter from the maize 16 kDaoleosin gene (OLE PRO), the coding sequence of soybean gamma-tocopherolmethyltransferase (SEQ ID NO:14) derived from cDNA clonesah1c.pk001.k8:fis (SEQ ID NO:13) (PCT Publication No. WO 00/032757) andthe polyadenylation signal sequence/terminator from the nopalinesynthase (NOS) gene of Agrobacterium tumefaciens is constructed usingmethods and technologies known in the art. A second expression cassettecomprises the barley HGGT coding sequence (PCT Publication No. WO03/082899; U.S. Application No. 2004/0034886) under the transcriptionalcontrol of the maize embryo abundant protein (EAP1) promoter andterminator, with the maize ADH1 INTRON1 inserted between the promoterand coding sequence for enhanced expression. The two expressioncassettes are linked, together with a gene encoding a selectable marker,in a binary vector suitable for Agrobacterium-mediated transformation ofmaize.

Similarly, a vector may be created as described above, with the maizegamma-tocopherol methyltransferase (SEQ ID NO:16) derived from cDNAclone p0060.coran49r:fis (SEQ ID NO:15) (PCT Publication No. WO00/032757) used in place of the soybean gamma-tocopherolmethyltransferase, using the same promoter/terminator elements and HGGTexpression cassette already described. Furthermore, one skilled in theart understands that the homogentisate geranylgeranyl transferases(HGGT) and gamma-tocopherol methyltransferases found in Table 1 andTable 2, respectively, may also be expressed in maize to demonstrate thefeasability of these cDNA for alpha and beta-tocotrienol production intransgenic plants.

An Agrobacterium-based protocol can be used for the transformation ofmaize (see below). The resulting binary vector is introduced intoAgrobacterium LBA4404 (PHP10523) cells, preferably by electroporation.An in vivo recombination generates a cointegrate plasmid between theintroduced binary vector and the vir plasmid (PHP10523) resident in theAgrobacterium cells. The resulting Agrobacterium cells are used totransform maize.

Transformation of Maize Mediated by Agrobacterium:

Freshly isolated immature embryos of maize, about ten days afterpollination (DAP), can be incubated with the Agrobacterium. Thepreferred genotype for transformation is the highly transformablegenotype Hi-II (Armstrong (1991) Maize Gen. Coop. Newsletter 65:92-93).An F1 hybrid created by crossing a Hi-II with an elite inbred may alsobe used. After Agrobacterium treatment of immature embryos, the embryoscan be cultured on medium containing toxic levels of herbicide. Onlythose cells that receive the herbicide resistance gene, and the linkedgene(s), grow on selective medium. Transgenic events so selected can bepropagated and regenerated to whole plants, produce seed, and transmittransgenes to progeny.

Preparation of Agrobacterium:

The engineered Agrobacterium tumefaciens LBA4404 can be constructed tocontain plasmids for seed-preferred expression of HGGT andgamma-tocopherol methyltransferase genes, as disclosed in U.S. Pat. No.5,591,616 (the contents of which are hereby incorporated by reference).To use the engineered construct in plant transformation, a master plateof a single bacterial colony transformed with plasmids forseed-preferred expression of HGGT and gamma-tocopherol methyltransferasegenes can be prepared by inoculating the bacteria on minimal AB mediumand allowing incubation at 28° C. for approximately three days. (Thecomposition and preparation of minimal AB medium has been previouslydescribed in PCT Publication No. WO 02/009040 (the contents of which arehereby incorporated by reference). A working plate can then be preparedby streaking the transformed Agrobacterium on YP medium (0.5% (w/v)yeast extract, 1% (w/v) peptone, 0.5% (w/v) sodium chloride, 1.5% (w/v)agar) that contains 50 μg/mL of spectinomycin.

The transformed Agrobacterium for plant transfection and co-cultivationcan then be prepared one day prior to maize transformation. Into 30 mLof minimal A medium (prepared as described in PCT Publication No. WO02/009040) in a flask was placed 50 μg/mL spectinomycin, 100 μMacetosyringone, and about a ⅛ loopful of Agrobacterium from a one totwo-day-old working plate. The Agrobacterium can then be grown at 28° C.with shaking at 200 rpm for approximately fourteen hours. At mid-logphase, the Agrobacterium can be harvested and resuspended at a densityof 3 to 5×108 CFU/mL in 561Q medium that contains 100 μM acetosyringoneusing standard microbial techniques. The composition and preparation of561Q medium was described in PCT Publication No. WO 02/009040.

Immature Embryo Preparation:

Nine to ten days after controlled pollination of a maize plant,developing immature embryos are opaque and 1-1.5 mm long. This length isthe optimal size for infection with the PHP18749-transformedAgrobacterium. The husked ears can be sterilized in 50% commercialbleach and one drop TWEEN®-20 for thirty minutes, and then rinsed twicewith sterile water. The immature embryos can then be aseptically removedfrom the caryopsis and placed into 2 mL of sterile holding solutionconsisting of medium 561Q that contains 100 μM of acetosyringone.

Agrobacterium Infection and Co-cultivation of Embryos:

The holding solution can be decanted from the excised immature embryosand replaced with transformed Agrobacterium. Following gentle mixing andincubation for about five minutes, the Agrobacterium can be decantedfrom the immature embryos. Immature embryos were then moved to a plateof 562P medium, the composition of which has been previously describedin PCT Publication No. WO 02/009040. The immature embryos can be placedon this media scutellum surface pointed upwards and then incubated at20° C. for three days in darkness. This can be followed by incubation at28° C. for three days in darkness on medium 562P that contains 100 μg/mLcarbenecillin as described in U.S. Pat. No. 5,981,840.

Selection of Transgenic Events:

Following incubation, the immature embryos can be transferred to 5630medium, which can be prepared as described in PCT Publication No. WO02/009040. This medium contains Bialaphos for selection of transgenicplant cells as conferred by the BAR gene that is linked to barley HGGTexpression cassette. At ten to fourteen-day intervals, embryos weretransferred to 5630 medium. Actively growing putative transgenicembryogenic tissue can be after six to eight weeks of incubation on the5630 medium.

Regeneration of T₀ Plants:

Transgenic embryogenic tissue is transferred to 288W medium andincubated at 28° C. in darkness until somatic embryos matured, or aboutten to eighteen days. Individual matured somatic embryos withwell-defined scutellum and coleoptile are transferred to 272 embryogermination medium and incubated at 28° C. in the light. After shootsand roots emerge, individual plants are potted in soil and hardened-offusing typical horticultural methods.

288W medium contains the following ingredients: 950 mL of deionizedwater; 4.3 g of MS Salts (Gibco); 0.1 g of myo-inositol; 5 mL of MSVitamins Stock Solution (Gibco); 1 mL of zeatin (5 mg/mL solution); 60 gsucrose; 8 g of agar (Sigma A-7049, Purified), 2 mL of indole aceticacid (0.5 mg/mL solution*); 1 mL of 0.1 mM ABA*; 3 mL of Bialaphos (1mg/mL solution*); and 2 mL of carbenicillin (50 mg/mL solution). The pHof this solution is adjusted to pH 5.6. The solution is autoclaved andingredients marked with an asterisk (*) are added after the media hascooled to 60° C.

Medium 272 contains the following ingredients: 950 mL of deionizedwater; 4.3 g of MS salts (Gibco); 0.1 g of myo-inositol; 5 mL of MSvitamins stock solution (Gibco); 40 g of Sucrose; and 1.5 g of GELRITE®.This solution is adjusted to pH 5.6 and then autoclaved.

Example 5 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant HGGT and gamma-tocopherolmethyltransferase polypeptides can be used to produce alpha- andbeta-tocotrienols in microbes such as algal and cyanobacterial cellsthat contain an operable tocopherol biosynthetic pathway. Expression ofcDNAs encoding the instant HGGT polypeptides in these cells are expectedto result in the condensation of geranylgeranyl pyrophosphate andhomogentisate. The product of the HGGT reaction2-methyl-6-geranylgeranylbenzoquinol can then be converted to alpha- andbeta-tocotrienols by tocopherol biosynthetic enzymes native to the hostmicrobial cell and the instant gamma-tocopherol methyltransferasepolypeptides. Tocotrienols can be produced in microbes by linking thecDNAs encoding the instant HGGT and gamma-tocopherol methyltransferasepolypeptides with promoter elements that are suitable to direct geneexpression in the selected host cell. The resulting chimeric genes canbe introduced into the host microbial cell using techniques such ashomologous recombination (Williams, J. G. K. (1988) Methods Enzymol.167:766-778; Legarde, D. et al. (2000) App. Environ. Microbiol.66:64-72). Host cells transformed with cDNAs for the instant HGGT andgamma-tocopherol methyltransferase polypeptides operably linked tofunctional promoters can then be analyzed for tocotrienol productionusing techniques described in Example 1.

Example 6 Production of Alpha- and Beta-Tocotrienol in Plant Cells

The cDNAs encoding the instant HGGT and gamma-tocopherolmethyltransferase polypeptides can be used to produce alpha- andbeta-tocotrienols in plant cells. Even higher levels of alpha- andbeta-tocotrienol production may be achieved when genes encoding theinstant HGGT and gamma-tocopherol methyltransferase polypeptides areco-expressed with genes that encode enzymes that participate either inthe conversion of plastidic chorismate pools to homogentisate or in theconversion of 2-methyl-6-prenylbenzoquinol to2,3-methyl-6-prenylbenzoquinol. To this end, transgenic plants aregenerated with DNA constructs that provide constitutive- orseed-specific expression of bifunctional chorismate mutase-prephenatedehydratase genes (TYRA) of bacterial or fungal origin andp-hydroxyphenylpyruvate dioxygenase genes (HPPD) and2-methyl-6-prenylbenzoquinol methyltransferase genes (VTE3) from plantsor photosynthetic bacteria. The TRYA gene products are targeted to thechloroplast by way of being fused to suitable chloroplast targetpeptides.

Plant transformations are performed as described above in Examples 1-3.Transgenic lines expressing high levels TYRA, HPPD and VTE3 areidentified by measuring tocochromanol content as described above inExamples 1-3. The events with high levels of tocochromanols are crossedto events generated with constructs expressing the instant HGGT andgamma-tocopherol methyltransferase polypeptides. Suitable constructs togenerate the latter events are KS319 (Example 1), SC1 and SC38 (Example2), KS270 and KS308 (Example 3). Alternatively, new DNA constructs aregenerated using standard methods of molecular biology that provideseed-specific or constitutive expression of five genes comprised ofTYRA, HPPD, VTE3 and HGGT and gamma-tocopherol methyltransferase genesof instant invention. Plant transformations are performed as describedin Examples 1-3. Transgenic lines expressing high levels of all fivegene products are identified by measuring tocochromanol content of planttissue as described in Examples 1-3.

Example 7 Production of Tocotrienols in Transgenic Soybean LinesMolecular Stack of Barley HGGT and Maize Gamma-TocopherolMethyltransferase

To demonstrate the ability to produce increased levels of alpha- andbeta-tocotrienols in transgenic soybean lines, the barley HGGT cDNA(bdl2c.pk006.o2; SEQ ID NO:2) and maize gamma-tocopherolmethyltransferase (p0060.coran49r:fis; SEQ ID NO:15) (PCT PublicationNo. WO 00/032757) were used in a molecular stack (progeny with bothtransgene-related traits).

A construct for seed specific expression of maize gamma-tocopherolmethyltransferase in soybean was generated as follows. DNA of KS126 (seeExample 1) was linearized with NotI. 5′ overhangs were completely filledin with T4 polynucleotid kinase and dephosphorylated using calfintestinal phosphatase. A restriction fragment containing the completeORF of the maize GTMT cDNA was excised from the EST clone usingrestriction enzymes DraI and SnaBI and ligated to the KS126 vector.Ligation products were introduced into E coli. Plasmid DNA was isolatedform recombinant clones and subjected to restriction digests with BamHI.Plasmid clones which produced a DNA fragment of 2.8 kb when digestedwith BamHI contain the maize GTMT gene in an orientation in which the 5′end of the transcript is in proximity to the 3′ end of the KTI promoter(sense orientation). This plasmid was named KS325. Its sequence is setforth as SEQ ID NO:51.

Transgenic soybean lines were generated with plasmid DNA of KS270 (seeExample 1) and KS325 using particle bombardment of embryogenic callus.

KS270 provides the barley HGGT gene under control of 617 bp of thesoybean β-conglycinin promoter. The polyadenylation signal for the HGGTtranscript is derived from the terminator of the phaseolin gene (fromthe bean Phaseolus vulgaris; Doyle et al. (1986) J. Biol. Chem.261:9228-9238). The plasmid also contains the cDNA of asulfonylurea-resistant variant of the soybean ALS gene that is undercontrol of 1217 bp of the SAMS promoter. The polyadenylation signal forthe HGGT transcript is derived from the terminator of the soybean ALSgene.

KS325 provides the gamma-tocopherol methyltransferase gene from maizeunder the control of 2090 bp of the soybean Kti promoter. Thepolyadeylation signal for the gamma-tocopherol methyltransferasetranscript is derived from the terminator of the Kti gene. KS325 alsoprovides a hygromycin B phosphotransferase (HPT) resistance gene (Gritzet al. (1983) Gene 25:179-188) that is under control of 1408 bp of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812). The polyadenylation signal for the hygromycin resistancegene is derived from the terminator of nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

Soybean embryogenic suspension cultures were transformed with DNAplasmids KS270 in conjunction with KS325 by the method of particle gunbombardment (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat.No. 4,945,050) using a BIORAD BIOLISTIC™ PDS1000/He instrument. Thefollowing stock solutions and media were used for transformation andregeneration of soybean plants:

Stock Solutions:

-   Sulfate 100× Stock: 37.0 g MgSO₄.7H₂O, 1.69 g MnSO₄.H₂O, 0.86 g    ZnSO₄.7H₂O, 0.0025 g CuSO₄.5H₂O-   Halides 100× Stock: 30.0 g CaCl₂.2H₂O, 0.083 g KI, 0.0025 g    CoCl₂.6H₂O-   P, B, Mo 100× Stock: 18.5 g KH₂PO₄, 0.62 g H₃BO₃, 0.025 g    Na₂MoO₄.2H₂O-   Fe EDTA 100× Stock: 3.724 g Na₂EDTA, 2.784 g FeSO₄.7H₂O-   2,4-D Stock: 10 mg/mL-   Vitamin B5 1000× Stock: 10.0 g myo-inositol, 0.10 g nicotinic acid,    0.10 g pyridoxine HCl, 1 g thiamine.    Media (per Liter):-   SB196: 10 mL of each of the above stock solutions, 1 mL B5 Vitamin    stock, 0.463 g (NH₄)₂SO₄, 2.83 g KNO₃, 1 mL 2,4-D stock, 1 g    asparagine, 10 g Sucrose, pH 5.7-   SB103: 1 pk. Murashige & Skoog salts mixture, 1 mL B5 Vitamin stock,    750 mg MgCl₂ hexahydrate, 60 g maltose, 2 g GELRITE®, pH 5.7.-   SB166: SB103 supplemented with 5 g per liter activated charcoal.-   SB71-4: Gamborg's B5 salts, 1 mL B5 vitamin stock, 30 g sucrose, 5 g    TC agar, pH 5.7.

To prepare tissue for transformation, soybean embryogenic suspensioncultures were maintained in 35 mL liquid medium (SB196) on a rotaryshaker (150 rpm) at 28° C. with fluorescent lights providing a 16 hourday/8 hour night cycle. Cultures were subcultured every two weeks byinoculating approximately 35 mg of tissue into 35 mL of fresh liquidmedia.

In particle gun bombardment procedures it is possible to use purified 1)entire plasmid DNA; or 2) DNA fragments containing only the recombinantDNA expression cassette(s) of interest. For every seventeen bombardmenttransformations, 85 μL of suspension is prepared containing 1 to 90picograms (pg) of plasmid DNA per base pair of each DNA plasmid. Bothrecombinant DNA plasmids were co-precipitated onto gold particles asfollows. The DNAs in suspension were added to 50 μL of a 20-60 mg/mL 0.6μm gold particle suspension and then combined with 50 μL CaCl₂ (2.5 M)and 20 μL spermidine (0.1 M). The mixture was vortexed for 5 seconds,spun in a microfuge for 5 seconds, and the supernatant removed. TheDNA-coated particles were then washed once with 150 μL of 100% ethanol,vortexed and spun in a microfuge again, then resuspended in 85 μL ofanhydrous ethanol. Five μL of the DNA-coated gold particles were thenloaded on each macrocarrier disk.

Approximately 150 to 250 mg of two-week-old suspension culture wasplaced in an empty 60 mm×15 mm petri plate and the residual liquidremoved from the tissue using a pipette. The tissue was placed about 3.5inches away from the retaining screen and each plate of tissue wasbombarded once. Membrane rupture pressure was set at 650 psi and thechamber was evacuated to −28 inches of Hg. Three plates were bombarded,and, following bombardment, the tissue from each plate was dividedbetween two flasks, placed back into liquid media, and cultured asdescribed above.

Seven days after bombardment, the liquid medium was exchanged with freshSB196 medium supplemented with 30-50 mg/L hygromycin. The selectivemedium was subsequently refreshed weekly or biweekly. Seven weekspost-bombardment, bright green, transformed tissue was observed growingfrom untransformed, chlorotic or necrotic embryogenic clusters. Isolatedgreen tissue was removed and inoculated into individual wells insix-well culture dishes to generate new, clonally-propagated,transformed embryogenic suspension cultures. Thus, each new line wastreated as independent transformation event in an individual well. Thesesuspensions can then be maintained as suspensions of embryos clusteredin an immature developmental stage through subculture or they can beregenerated into whole plants by maturation and germination ofindividual somatic embryos.

After two weeks in individual cell wells, transformed embryogenicclusters were removed from liquid culture and placed on solidifiedmedium (SB166) containing no hormones or antibiotics for one week.Embryos were cultured for at 26° C. with mixed fluorescent andincandescent lights on a 16 hour day/8 hour night schedule. After oneweek, the cultures were then transferred to SB103 medium and maintainedin the same growth conditions for 3 additional weeks.

Somatic embryos became suitable for germination after four weeks andwere then removed from the maturation medium and dried in empty petridishes for 1 to 5 days. The dried embryos were then planted in SB71-4medium where they were allowed to germinate under the same light andtemperature conditions as described above. Germinated embryos weretransferred to sterile soil and grown to maturity for seed production.

A total of eighteen events were created by co-transformation with KS270and KS325 plasmids. Tocol composition of five T1 seed was assayed foreach events as follows. A seed chip (approximately 5-15 mg of tissue)was obtained from the cotyledon tissue of the seed. The chip wasextracted with 100 μL of heptane for 2 hours. Tocopherol and tocotrienolwas quantitated by HPLC analysis as described in Example 3.

A total of eighteen events were generated and analyzed (see Table 13).

TABLE 13 Tocol Composition (percent of total tocopherols (tocph.) andtocotrienols (toct.)) for T1 Seed Chips of Events Generated with KS270and KS325 alpha- beta- gamma- delta- alpha- beta- gamma- delta- tocph.toct. Event ID tocph. tocph. tocph. tocph. toct. toct. toct toct. (ppm)(ppm) 4652.1.10.1A 1 1 2 3 2 1 44 46 167 2175 4652.1.10.1B 17 2 80 0 0 00 0 240 0 4652.1.10.1C 2 1 3 0 2 1 44 47 279 4711 4652.1.10.1D 11 1 88 00 0 0 0 310 0 4652.1.10.1E 2 1 4 0 4 2 42 46 317 4070 4652.1.11.1A 1 0 30 0 0 44 51 156 3463 4652.1.11.1B 1 1 4 0 0 0 47 47 127 21334652.1.11.1C 1 0 4 0 0 0 50 46 96 1900 4652.1.11.1D 20 2 78 0 0 0 0 0270 0 4652.1.11.1E 1 0 5 0 0 0 53 39 201 2600 4652.1.2.1A 1 0 3 0 1 0 4252 101 2204 4652.1.2.1B 1 0 3 0 1 0 46 50 149 3923 4652.1.2.1C 11 1 88 00 0 0 0 192 0 4652.1.2.1D 1 0 3 0 1 0 44 51 153 3310 4652.1.2.1E 0 0 3 01 0 41 54 109 2831 4652.1.7.1A 6 4 0 0 38 41 4 7 240 2051 4652.1.7.1B 64 0 0 42 40 3 5 169 1597 4652.1.7.1C 22 2 76 0 0 0 0 0 273 0 4652.1.7.1D6 5 0 0 35 45 3 6 214 1756 4652.1.7.1E 5 5 0 0 32 52 2 5 400 36704652.1.8.1A 16 2 83 0 0 0 0 0 175 0 4652.1.8.1B 0 0 4 0 0 0 47 48 1152429 4652.1.8.1C 17 2 82 0 0 0 0 0 160 0 4652.1.8.1D 1 0 4 0 0 0 45 50114 2277 4652.1.8.1E 1 0 4 0 0 0 51 44 100 1962 4652.2.10.1A 0 0 4 0 0 042 53 124 2292 4652.2.10.1B 0 0 4 0 0 0 47 47 147 2767 4652.2.10.1C 1 05 0 0 0 48 46 223 3502 4652.2.10.1D 9 1 90 0 0 0 0 0 254 0 4652.2.10.1E11 2 87 0 0 0 0 0 267 0 4652.2.11.1A 11 1 87 0 0 0 0 0 164 04652.2.11.1B 6 5 0 0 37 50 0 1 197 1604 4652.2.11.1C 7 6 0 0 36 50 0 0466 2950 4652.2.11.1D 12 1 86 0 0 0 0 0 209 0 4652.2.11.1E 6 7 0 0 32 550 0 440 2973 4652.2.13.1A 10 1 88 0 0 0 0 0 243 0 4652.2.13.1B 10 1 88 00 0 0 0 230 0 4652.2.13.1C 15 2 82 0 0 0 0 0 155 0 4652.2.13.1D 11 1 880 0 0 0 0 284 0 4652.2.13.1E 11 1 88 0 0 0 0 0 229 0 4652.2.14.1A 85 141 0 0 0 0 0 360 0 4652.2.14.1B 4 4 0 0 31 43 5 12 267 2796 4652.2.14.1C9 6 0 0 40 44 0 1 342 1855 4652.2.14.1D 86 13 0 0 0 0 0 0 254 14652.2.14.1E 5 4 0 0 32 58 0 1 262 2495 4652.2.6.1A 6 8 0 0 32 54 0 0353 2192 4652.2.6.1B 65 14 0 0 15 6 0 0 378 102 4652.2.6.1C 8 7 0 0 3352 0 0 488 2762 4652.2.6.1D 6 6 0 0 33 53 0 1 399 2905 4652.2.6.1E 63 160 0 15 6 0 0 358 95 4652.2.7.1A 2 1 4 0 1 1 42 49 205 2779 4652.2.7.1B 21 4 0 2 1 43 48 176 2660 4652.2.7.1C 1 1 3 0 2 1 45 48 110 21924652.2.7.1D 1 1 4 0 2 1 42 50 170 2679 4652.2.7.1E 3 1 6 0 2 1 48 40 1991889 4652.2.9.1A 5 4 0 0 28 31 11 22 252 2495 4652.2.9.1B 6 5 0 0 33 405 11 214 1614 4652.2.9.1C 4 2 3 0 17 8 37 29 212 2148 4652.2.9.1D 5 4 00 30 33 10 19 245 2521 4652.2.9.1E 4 2 1 0 19 14 24 36 194 22124652.3.15.1A 85 14 1 0 0 0 0 0 213 0 4652.3.15.1B 76 23 0 0 0 0 0 0 3790 4652.3.15.1C 13 2 86 0 0 0 0 0 183 0 4652.3.15.1D 77 22 0 0 0 0 0 1167 1 4652.3.15.1E 78 21 1 0 0 0 0 0 248 0 4652.3.17.1A 8 7 0 0 36 47 11 361 2029 4652.3.17.1B 8 5 0 0 42 44 1 1 362 2419 4652.3.17.1C 18 10 00 34 37 0 1 471 1198 4652.3.17.1D 8 6 0 0 38 45 1 1 334 19414652.3.17.1E 9 7 0 0 38 45 0 1 276 1392 4652.3.3.1A 4 4 0 0 37 41 5 9272 2905 4652.3.3.1B 5 4 0 0 37 45 3 6 282 2714 4652.3.3.1C 8 6 0 0 3648 1 2 416 2608 4652.3.3.1D 4 4 0 0 36 53 1 2 233 2390 4652.3.3.1E 5 5 00 31 43 5 12 344 3319 4652.3.5.1A 18 2 80 0 0 0 0 0 161 0 4652.3.5.1B 212 77 0 0 0 0 0 192 0 4652.3.5.1C 4 4 0 0 22 28 13 29 203 23154652.3.5.1D 18 2 80 0 0 0 0 0 191 0 4652.3.5.1E 6 4 1 0 28 27 14 20 2962450 4652.3.6.1A 16 2 82 0 0 0 0 0 182 0 4652.3.6.1B 7 5 0 0 43 43 1 1328 2451 4652.3.6.1C 7 5 0 0 41 44 1 1 292 2060 4652.3.6.1D 9 6 0 0 4142 1 1 288 1654 4652.3.6.1E 15 2 84 0 0 0 0 0 244 0 4652.3.8.1A 30 4 660 0 0 0 0 137 0 4652.3.8.1B 24 3 73 0 0 0 0 0 180 0 4652.3.8.1C 16 2 820 0 0 0 0 196 0 4652.3.8.1D 30 3 68 0 0 0 0 0 205 0 4652.3.8.1E 44 6 490 0 0 0 0 194 0

Seed chips from fifteen events contained significant levels oftocotrienol. Ten of these also contained significant levels (>150 ppm)of alpha- and beta-tocotrienol. Alpha-tocotrienol content in seed chipsreached 1300 ppm in event 4652.1.7.1E (i.e, (400+3670)×0.32=1302). Forseveral events greater than 40% of the total tocopherol and tocotrienolcontent was alpha-tocotrienol. Seed chips do not provide a comprehensivepicture of the oil composition of the entire seed. Therefore, the entireT1 seed from selected events were subjected to tocol analysis asdescribed in Example 2 (see Table 14).

TABLE 14 Tocol Composition (percent of total tocopherols (tocph.) andtocotrienols (toct.)) for T1 Seed of Events Generated with KS270 andKS325 alpha- beta- gamma- delta- alpha- beta- gamma- delta- tocph. toct.Event ID tocph. tocph. tocph. tocph. toct. toct. toct toct. (ppm) (ppm)4652.1.7.1 A 5 4 0 0 31 45 3 10 261 2355 4652.1.7.1 B 5 4 0 0 36 44 3 8214 2162 4652.2.11.1 B 4 5 0 0 29 59 1 2 213 2028 4652.2.11.1 C 6 7 0 024 62 0 0 224 1414 4652.2.14.1 C 6 6 0 0 30 53 1 3 245 1694 4652.2.6.1 C7 9 0 0 25 57 0 2 320 1636 4652.2.6.1 D 7 8 0 0 27 57 0 1 327 19864652.3.17.1 B 5 6 0 0 28 54 2 6 210 1688 4652.3.17.1 D 7 6 0 0 31 51 1 4238 1612 4652.3.6.1 B 5 4 0 0 36 51 1 2 227 2077 4652.3.6.1 C 6 5 0 0 3550 1 2 238 1802

The highest whole seed alpha-tocotrienol level (847 ppm) was reached inevent 4652.1.7.1. For the six events subjected to whole seed tocolanalysis at least 24% and up to 36% of the total tocopherol andtocotrienol content was derived from alpha-tocotrienol. In all sixevents gamma- and delta-tocotrienol levels are at very low levelscompared to the best transgenic event generated in similar experimentsperformed with the soybean GTMT sequence (Example 2). The maize GTMTprovides an excellent enzyme for methylation of gamma- anddelta-tocotrienol in developing soybean seed.

Example 8 Alpha-Tocotrienol Production in Arabidopsis thaliana byTransgenic Expression of Barley HGGT and Maize Gamma-TocopherolMethyltransferase

A construct for co-expression of barley homogentisate geranylgeranyltransferase and maize gamma-tocopherol methyltransferase in Arabidopsisthaliana was generated as follows. The maize GTMT expression cassettecomprised of Kti promoter GTMT gene and Kti terminator was excised fromKS325 (see Example 7) as a 3.6 kb fragment by complete digestion withAscI. This DNA fragment was ligated to SC38 DNA that had previously beenlineraized by partial digestion with AscI. Recombinant clones wererecovered and plasmid DNA was isolated using standard techniques. Thisnew plasmid is referred to KS325×SC38. A 6.7 kb DNA fragment containingexpression cassettes for barley HGGT and maize GTMT genes was excisedfrom this plasmid by partial digestion with SalI and ligated to pZBL120(see Example 1) linearized with SalI to give pZBL120×KS325×SC38. TheT-DNA of the plant transformation vector pZBL120×KS325×SC38 is set forthas SEQ ID NO:52. Transgenic Arabidopsis lines were generated usingpZBL20×KS325×SC38 as described in Example 1. A total of 38 lines weregenerated and tocochromanol content of T2 seed was determined by HPLCanalysis as described in Example 1 (see Table 15).

TABLE 15 Tocol Composition (percent of total tocopherols (tocph.) andtocotrienols (toct.)) of T2 Seed Material of Transgenic ArabidopsisLines Expressing Barley HGGT and Maize Gamma-TocopherolMethyltransferase Genes alpha- beta- gamma- delta- alpha- beta- gamma-delta- tocph. toct. Event ID tocph. tocph. tocph. tocph. toct. toct.toct toct. (ppm) (ppm) 38 24 1 14 1 51 2 7 1 244 382 17 33 1 12 0 50 0 40 327 382 31 27 1 14 1 49 0 8 1 421 577  3 30 0 14 0 49 0 7 0 489 626 3424 1 12 1 49 3 9 2 180 300 32 32 1 12 1 49 2 4 0 347 418  2 28 1 18 1 480 3 0 254 271 35 24 1 15 1 48 2 8 1 245 348  6 23 0 30 0 47 0 0 0 165148 12 17 2 7 1 47 7 16 2 388 987 29 26 1 13 1 47 2 8 2 318 461 25 29 114 1 47 2 6 1 327 407 15 25 1 22 1 47 2 2 0 350 374 18 27 1 16 1 46 0 81 344 429 27 26 1 16 1 45 2 8 1 335 435 33 28 1 16 1 45 0 7 1 214 246 2029 1 16 1 45 0 8 1 330 385 13 28 0 17 1 44 0 9 1 356 419 26 27 1 20 1 400 11 1 284 312 30 35 1 17 1 40 0 7 1 400 354 21 31 0 22 1 38 0 8 1 329282 22 14 1 11 1 38 3 29 4 358 965  1 38 0 21 1 34 0 6 0 422 286  5 31 028 0 33 0 8 0 168 117 28 49 1 19 0 29 0 2 0 240 108 10 22 0 39 1 29 0 91 260 160 11 3 0 94 1 2 0 0 0 377 8 23 69 0 30 1 0 0 0 0 400 2  4 1 0 981 0 0 0 0 291 0  7 17 0 82 1 0 0 0 0 311 0  8 1 0 98 1 0 0 0 0 347 0  91 0 98 2 0 0 0 0 417 0 14 65 0 34 1 0 0 0 0 426 0 16 1 0 98 2 0 0 0 0266 0 19 1 0 98 1 0 0 0 0 379 0 24 68 1 30 1 0 0 0 0 323 0 36 69 1 30 10 0 0 0 305 0 37 1 0 97 2 0 0 0 0 262 0 wild-type 1 0 98 1 0 0 0 0 173 0

Of the 38 events analyzed 26 showed greater than 100 ppm tocotrienolsand reached levels as high as 990 ppm. In these 26 eventsalpha-tocotrienol represented at least 28% and as much as 51% of thetotal tocochromanol content. In T2 seed of the best event (Event ID 12)alpha-tocotrienol levels reached 640 ppm (i.e., (388+987)×0.47=646). TheT2 material described so far still contains 25% of wild-type seed.Events 3, 12, 29, 31 and 32 were germinated on selective media. Whengrown on selective media T2 seed of all six events produced 25% ofkanamycin-sensitive wild-type seed. For each event 15 kanamycinresistant seedlings were transferred to soil allowed to self-fertilizeand grown to maturity. For each event three T3 seed selections wereidentified that no longer segregated kanamycin-sensitive seedlings. Thisseed material was subjected to tocochromanol quantitation as describedabove (see Table 16).

TABLE 16 Tocol Composition (percent of total tocopherols (tocph.) andtocotrienols (toct.)) of Homozygous T3 Seed Material of TransgenicArabidopsis Lines Expressing Barley HGGT and Maize Gamma-TocopherolMethyltransferase Genes alpha- beta- gamma- delta- alpha- beta- gamma-delta- tocph. toct. Event ID tocph. tocph. tocph. tocph. toct. toct.toct toct. (ppm) (ppm) 3 29 1 2 1 61 2 3 1 229 464 3 33 1 2 1 58 2 3 0493 849 3 32 1 2 1 58 2 4 1 307 545 12 24 2 2 1 59 7 4 1 407 971 12 19 51 1 55 14 4 2 361 1031  12 21 3 9 1 53 7 5 1 441 880 29 23 2 2 0 58 6 72 345 943 29 28 2 3 0 53 4 8 2 320 647 29 17 2 2 0 51 7 15 5 219 770 3221 2 1 0 64 8 4 1 213 675 32 22 2 1 0 63 6 4 1 291 841 32 24 2 2 1 61 54 1 346 865 31 21 2 2 2 65 3 4 1 300 795 31 22 3 1 1 66 4 3 1 213 562 3121 3 2 2 63 4 5 1 297 785

In the homozygous T3 seed material of the five events eventsalpha-tocotrienol represented at least 51% and as much as 65% of thetotal tocochromanol content. In homozygous T3 seed of one event (EventID 12) alpha-tocotrienol levels reached 810 ppm (i.e.,(407+971)×0.59=813). In all five events gamma tocotrienol levels are atvery low levels compared to the best transgenic event generated insimilar experiments performed with the soybean GTMT sequence (Example1). The maize GTMT provides an excellent enzyme for methylation ofgamma-tocotrienol in developing Arabidopsis seed.

Example 9 Preparation of cdna Libraries and Isolation and Sequencing ofcdna Clones

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in UNI-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The UNI-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBLUESCRIPT®. In addition, thecDNAs may be introduced directly into precut Bluescript® II SK(+)vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followedby transfection into DH10B cells according to the manufacturer'sprotocol (GIBCO BRL Products). Once the cDNA inserts are in plasmidvectors, plasmid DNAs are prepared from randomly picked bacterialcolonies containing recombinant pBLUESCRIPT® plasmids, or the insertcDNA sequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

Confirmed templates are transposed via the Primer Island transpositionkit (PE Applied Biosystems, Foster City, Calif.) which is based upon theSaccharomyces cerevisiae Ty1 transposable element (Devine and Boeke(1994) Nucleic Acids Res. 22:3765-3772). The in vitro transpositionsystem places unique binding sites randomly throughout a population oflarge DNA molecules. The transposed DNA is then used to transform DH10Belectro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.)via electroporation. The transposable element contains an additionalselectable marker (named DHFR; Fling and Richards (1983) Nucleic AcidsRes. 11:5147-5158), allowing for dual selection on agar plates of onlythose subclones containing the integrated transposon. Multiple subclonesare randomly selected from each transposition reaction, plasmid DNAs areprepared via alkaline lysis, and templates are sequenced (ABI PRISM®dye-terminator ReadyReaction mix) outward from the transposition eventsite, utilizing unique primers specific to the binding sites within thetransposon.

Sequence data is collected (ABI PRISM® Collections) and assembled usingPhred and Phrap (Ewing et al. (1998) Genome Res. 8:175-185; Ewing andGreen (1998) Genome Res. 8:186-194). Phred is a public domain softwareprogram which re-reads the ABI sequence data, re-calls the bases,assigns quality values, and writes the base calls and quality valuesinto editable output files. The Phrap sequence assembly program usesthese quality values to increase the accuracy of the assembled sequencecontigs. Assemblies are viewed by the Consed sequence editor (Gordon etal. (1998) Genome Res. 8:195-202).

In some of the clones the cDNA fragment may correspond to a portion ofthe 3′-terminus of the gene and does not cover the entire open readingframe. In order to obtain the upstream information one of two differentprotocols is used. The first of these methods results in the productionof a fragment of DNA containing a portion of the desired gene sequencewhile the second method results in the production of a fragmentcontaining the entire open reading frame. Both of these methods use tworounds of PCR amplification to obtain fragments from one or morelibraries. The libraries some times are chosen based on previousknowledge that the specific gene should be found in a certain tissue andsome times are randomly-chosen. Reactions to obtain the same gene may beperformed on several libraries in parallel or on a pool of libraries.Library pools are normally prepared using from 3 to 5 differentlibraries and normalized to a uniform dilution. In the first round ofamplification both methods use a vector-specific (forward) primercorresponding to a portion of the vector located at the 5′-terminus ofthe clone coupled with a gene-specific (reverse) primer. The firstmethod uses a sequence that is complementary to a portion of the alreadyknown gene sequence while the second method uses a gene-specific primercomplementary to a portion of the 3′-untranslated region (also referredto as UTR). In the second round of amplification a nested set of primersis used for both methods. The resulting DNA fragment is ligated into apBLUESCRIPT® vector using a commercial kit and following themanufacturer's protocol. This kit is selected from many available fromseveral vendors including INVITROGEN™ (Carlsbad, Calif.), PromegaBiotech (Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmidDNA is isolated by alkaline lysis method and submitted for sequencingand assembly using Phred/Phrap, as above.

Example 10 Identification of cDNA Clones

cDNA clones encoding ferrochelatases can be identified by conductingBLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol.Biol. 215:403-410; see also the explanation of the BLAST algorithm onthe world wide web site for the National Center for BiotechnologyInformation at the National Library of Medicine of the NationalInstitutes of Health) searches for similarity to amino acid sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). The DNAsequences from clones can be translated in all reading frames andcompared for similarity to all publicly available protein sequencescontained in the “nr” database using the BLASTX algorithm (Gish andStates (1993) Nat. Genet. 3:266-272) provided by the NCBI. Thepolypeptides encoded by the cDNA sequences can be analyzed forsimilarity to all publicly available amino acid sequences contained inthe “nr” database using the BLASTP algorithm provided by the NationalCenter for Biotechnology Information (NCBI). For convenience, theP-value (probability) or the E-value (expectation) of observing a matchof a cDNA-encoded sequence to a sequence contained in the searcheddatabases merely by chance as calculated by BLAST are reported herein as“pLog” values, which represent the negative of the logarithm of thereported P-value or E-value. Accordingly, the greater the pLog value,the greater the likelihood that the cDNA-encoded sequence and the BLAST“hit” represent homologous proteins.

ESTs sequences can be compared to the Genbank database as describedabove. ESTs that contain sequences more 5- or 3-prime can be found byusing the BLASTn algorithm (Altschul et al (1997) Nucleic Acids Res.25:3389-3402.) against the Du Pont proprietary database comparingnucleotide sequences that share common or overlapping regions ofsequence homology. Where common or overlapping sequences exist betweentwo or more nucleic acid fragments, the sequences can be assembled intoa single contiguous nucleotide sequence, thus extending the originalfragment in either the 5 or 3 prime direction. Once the most 5-prime ESTis identified, its complete sequence can be determined by Full InsertSequencing as described above. Homologous genes belonging to differentspecies can be found by comparing the amino acid sequence of a knowngene (from either a proprietary source or a public database) against anEST database using the tBLASTn algorithm. The tBLASTn algorithm searchesan amino acid query against a nucleotide database that is translated inall 6 reading frames. This search allows for differences in nucleotidecodon usage between different species, and for codon degeneracy.

Example 11 Characterization of a cDNA Clones Encoding2-Methyl-6-Phytylbenzoquinol Methyltransferase

A cDNA library representing mRNAs from developing seed tissue of balsampear (Momordica charantia) was prepared and a cDNA clone,fds1n.pk003.e5, was identified that encodes 2-methyl-6-phytylbenzoquinolmethyltransferase (MC VTE3). The nucleic acid sequence of theprotein-coding region of the cDNA insert in fds1n.pk003.e5 is presentedas SEQ ID NO:53. The amino acid sequence of the protein encoded by SEQID NO:53 is presented as SEQ ID NO:54. The amino acid sequence of theputative mature protein, i.e., minus the transit peptide (amino acids1-47 of SEQ ID NO:54), is presented as SEQ ID NO:70.

Shown in Table 17 are the BLASTP results, expressed as pLog of theE-value, for SEQ ID NO:54 and each of the indicated polypeptides.Polypeptides in which the putative transit peptide has been removed areindicated as “mature”. The amino acid sequence of the mature Arabidopsis2-methyl-6-phytylbenzoquinol methyltransferase polypeptide (SEQ IDNO:67) is taken from Cheng et al. 2003 Plant Cell 15:2343-2356. Alsoshown in Table 17 are the percent sequence identity values for betweenSEQ ID NO:54 and each of the indicated amino acid sequences:

TABLE 17 BLAST Results and Percent Sequence Identity for the2-Methyl-6-Phytylbenzoquinol Methyltransferase From Momordica charantia(SEQ ID NO: 54) NCBI GI No. BLASTP Percent or Patent SEQ ID pLog ofSequence Reference Plant NO E-value Identity 108385436 Arabidopsis 61154 80.7 157348021 Grape 62 168 83.0 80/971,672 Sunflower 63 162 81.8US2007061916 Cotton 64 171 86.0 WO2003034812 Soybean 65 168 84.7WO2003034812 Corn 66 141 73.1 108385436-derived Arabidopsis 67 — 88.9(mature) WO2003034812 Soybean 68 162 92.4 (mature) WO2003034812 Corn 69139 80.2 (mature)

SEQ ID NO:70 is the amino acid sequence of the putative mature2-methyl-6-phytylbenzoquinol methyltransferase from Momordica charantia.Shown in Table 18 are the percent sequence identity values between SEQID NO:70 and each of the indicated amino acid sequences:

TABLE 18 Percent Sequence Identity with the Mature 2-Methyl-6-Phytylbenzoquinol Methyltransferase From Momordica charantia(SEQ ID NO: 70) NCBI GI No. Percent or Patent SEQ ID Sequence ReferencePlant NO Identity 108385436 Arabidopsis 61 87.8 157348021 Grape 62 88.580/971,672 Sunflower 63 89.6 US2007061916 Cotton 64 92.0 WO2003034812Soybean 65 92.4 WO2003034812 Corn 66 80.2 108385436-derived Arabidopsis(mature) 67 88.9 WO2003034812 Soybean (mature) 68 92.4 WO2003034812 Corn(mature) 69 80.2

FIGS. 3A-3C present an alignment of the amino acid sequences of the2-methyl-6-phytylbenzoquinol methyltransferase proteins set forth in SEQID NOs: 54, 61, 62, 63, 64, 65, 66, 67, 68, 69 and 70. FIG. 4 presentsthe percent sequence identities and divergence values for each sequencepair presented in FIGS. 3A-3C.

Sequence alignments and percent identity calculations were performedusing the MEGALIGN® program of the LASERGENE® bioinformatics computingsuite (DNASTAR® Inc., Madison, Wis.). Alignment of the sequences wasperformed using the Clustal W method of alignment wih the defaultparameters. Default parameters for multiple alignments were GapPenalty=10, Gap Length Penalty=0.20, Delay Divergent Sequence=30%, andDNA Transition Weight=0.50. Default parameters for pairwise alignmentswere Gap Penalty=10.0 and Gap Length=0.10.

Example 12 Tocol Composition of Soybean Somatic Embryos Transformed withBarley HGGT, Maize Gamma-Tocopherol Methyltransferase and Momordicacharantia 2-Methyl-6-Phytylbenzoquinol Methyltransferase

EST clone fds1n.pk003.e5 is derived from a cDNA library of developingseed tissue of balsam pear (Momordica charantia) encodes a protein with83% sequence identity to the VTE3 gene product of Arabidopsis (PlantCell (2003), 15(10), 2343-2356), using the CLUSTAL W method ofalignment. The DNA sequence of the open-reading frame in the cDNA insertis set forth as SEQ ID NO:53. The predicted amino acid sequence of theMomordica charantia 2-methyl-6-phytylbenzoquinol methyltransferase,designated “MC VTE3”, is set forth as SEQ ID NO:54. A DNA fragment wasgenerated by PCR. The ORF was amplified from plasmid DNA usingoligonucleotide primers. The sequences of the sense (forward) andantisense (reverse) oligonucleotide primers used in this reaction wereas follows:

(SEQ ID NO: 55) 5′-CACCATGGCTTCTGCAATGCTCAATGG -3′ and (SEQ ID NO: 56)5′-CTCCCCAACTCAGATTGGTTGCCCTTC-3′.PCR amplification was achieved using TAQ polymerase, and plasmid DNA ofthe EST clone was used as the template. The product of this PCR reactionwas purified by agarose gel electrophoresis and subcloned intopENTR/D-TOPO® (INVITROGEN™) as described in the manufacturer's protocol.A 1042 bp fragment containing the entire open-reading frame of wasexcised using restriction enzymes AscI and NotI. Ends were completelyfilled in with T4 polymerase (INVITROGEN™, USA) according toinstructions of the manufacturer and ligated to NotI linearized,filled-in pKR561 vector. Recombinant clones were subjected to analysisby restriction enzyme digestion to identify ligation products in whichthe start codon was in proximity of the annexin promoter in pKR561(sense orientation). Plasmids with this orientation are henceforthreferred to as pKR561-MCVTE3 (SEQ ID NO:57).

Vector pKR561 had previously been constructed as follows. Vector pKR268(SEQ ID NO:58), which was previously described in U.S. Pat. No.7,256,033 (the contents of which are hereby incorporated by reference),contains a NotI site flanked by the soybean annexin promoter (U.S. Pat.No. 7,129,089) and the BD30 3′ termination region (Ann/NotI/BD30cassette). Vector pKR145 (SEQ ID NO:59), which was previously describedin PCT Publication No. WO 2004/071467 (the contents of which are herebyincorporated by reference), contains the hygromycin B phosphotransferasegene [Gritz, L. and Davies, J. (1983) Gene 25:179-188], flanked by theT7 promoter and transcription terminator (T7prom/hpt/T7term cassette),and a bacterial origin of replication (ori) for selection andreplication in E. coli. In addition, pKR145 contains the hygromycin Bphosphotransferase gene, flanked by the 35S promoter [Odell et al.,(1985) Nature 313:810-812] and NOS 3′ transcription terminator [Depickeret al., (1982) J. Mol. Appl. Genet. 1:561:570] (35S/hpt/NOS3′ cassette)for selection in soybean. The BsiWI fragment of pKR268, containing theAnn/NotI/BD30 cassette, was cloned into the BsiWI fragment of pKR145,containing the 35S/hpt/NOS3′ cassette), to produce pKR561 (SEQ IDNO:60).

Generation Of Transgenic Somatic Embryos:

For co-expression of HV HGGT, ZM GTMT (VTE4) and MC VTE3 genes insoybean somatic embryos, soybean tissue was co-bombarded as describedbelow with a mixture of KS325×SC38 (see Example 8) and pKR561-MCVTE3.Prior to mixing the DNAs, KS325×SC38 was digested with EcoRI and BglIIto inactivate vector components conferring hygromycin resistance.Likewise pKR561-MCVTE3 was linearized with BamHI. KS325×SC38 andpKR561-MCVTE3 were combined in a 10:1 ratio and used for transformationof soybean somatic embryos as described below. In the resulting DNAmixture the linearized pKR561-MCVTE3 DNA fragment provides an intactexpression cassette for hygromycin resistance comprised of CaMV 35Spromoter hygromycin phosphotransferase gene and nos terminator.

Culture Conditions:

Soybean embryogenic suspension cultures (cv. Jack) were maintained in 35mL liquid medium SB196 (infra) on a rotary shaker, 150 rpm, 26° C. withcool white fluorescent lights on 16:8 h day/night photoperiod at lightintensity of 60-85 μE/m2/s. Cultures were subcultured every 7 days totwo weeks by inoculating approximately 35 mg of tissue into 35 mL offresh liquid SB196 (the preferred subculture interval is every 7 days).

Soybean embryogenic suspension cultures were transformed with thesoybean expression plasmids by the method of particle gun bombardment(Klein et al., Nature 327:70 (1987)) using a DUPONT™ BIOLISTIC™PDS1000/HE instrument (helium retrofit) for all transformations.

Soybean Embryogenic Suspension Culture Initiation:

Soybean cultures were initiated twice each month with 5-7 days betweeneach initiation. Pods with immature seeds from available soybean plants45-55 days after planting were picked, removed from their shells andplaced into a sterilized magenta box. The soybean seeds were sterilizedby shaking them for 15 min in a 5% Clorox solution with 1 drop of ivorysoap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1drop of soap, mixed well). Seeds were rinsed using two 1-liter bottlesof sterile distilled water and those less than 4 mm were placed onindividual microscope slides. The small end of the seed was cut and thecotyledons pressed out of the seed coat. Cotyledons were transferred toplates containing SB199 medium (25-30 cotyledons per plate) for 2 weeks,then transferred to SB1 for 2-4 weeks. Plates were wrapped with fibertape. After this time, secondary embryos were cut and placed into SB196liquid media for 7 days.

Preparation of DNA for Bombardment:

Either an intact plasmid or a DNA plasmid fragment containing the genesof interest and the selectable marker gene were used for bombardment.

A 50 μL aliquot of sterile distilled water containing 1 mg of goldparticles was added to 5 μL of a 1 μg/μL DNA solution (DNA fragmentsprepared as described above), 50 μL 2.5M CaCl₂ and 20 μL of 0.1 Mspermidine. The mixture was pulsed 5 times on level 4 of a vortex shakerand spun for 5 sec in a bench microfuge. After a wash with 150 μL of100% ethanol, the pellet was suspended by sonication in 85 μL of 100%ethanol. Five μL of DNA suspension was dispensed to each flying disk ofthe BIOLISTIC™ PDS1000/HE instrument disk. Each 5 μL aliquot containedapproximately 0.058 mg gold particles per bombardment (i.e., per disk).

Tissue Preparation and Bombardment with DNA:

Approximately 100-150 mg of 7 day old embryonic suspension cultures wereplaced in an empty, sterile 60×15 mm petri dish and the dish was placedinside of an empty 150×25 mm Petri dish. Tissue was bombarded 1 shot perplate with membrane rupture pressure set at 650 PSI and the chamber wasevacuated to a vacuum of 27-28 inches of mercury. Tissue was placedapproximately 2.5 inches from the retaining/stopping screen.

Selection of Transformed Embryos:

Transformed embryos were selected using hygromycin as the selectablemarker. Specifically, following bombardment, the tissue was placed intofresh SB196 media and cultured as described above. Six to eight dayspost-bombardment, the SB196 is exchanged with fresh SB196 containing 30mg/L hygromycin. The selection media was refreshed weekly. Four to sixweeks post-selection, green, transformed tissue was observed growingfrom untransformed, necrotic embryogenic clusters. Isolated, greentissue was removed and inoculated into multi-well plates to generatenew, clonally propagated, transformed embryogenic suspension cultures.

Embryo Maturation:

Transformed embryogenic clusters were cultured for one-three weeks at26° C. in SB196 under cool white fluorescent (Phillips cool whiteEconowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) ona 16:8 hr photoperiod with light intensity of 90-120 μE/m²s. After thistime embryo clusters were removed to a solid agar media, SB166, for 1week, then subcultured to medium SB103 for 3 weeks. Alternatively,embryo clusters were removed to SB228 (SHaM) liquid media, 35 mL in 250mL Erlenmeyer flask, for 2-3 weeks. Tissue cultured in SB228 wasmaintained on a rotary shaker, 130 rpm, 26° C. with cool whitefluorescent lights on 16:8 h day/night photoperiod at light intensity of60-85 μE/m2/s. During this period, individual embryos were removed fromthe clusters and screened for alterations in their fatty acidcompositions as described supra.

Media Recipes:

SB196-FN Lite Liquid Proliferation Medium (per liter) contains thefollowing: 10 mL of MS FeEDTA-100× Stock 1; 10 mL of MS Sulfate-100×Stock 2; 10 mL of FN Lite Halides-100× Stock 3; 10 mL of FN Lite P, B,Mo-100× Stock 4; 1.0 mL of B5 vitamins (1 mL/L); 1.0 mL of 2,4-D (10mg/L final concentration); 2.83 g of KNO₃; 0.463 g of (NH₄)₂SO₄; 1.0 gof Asparagine; 10 g of Sucrose (1%); adjust to pH 5.8.

FN Lite Stock Solutions No. 1-4 are prepared as follows:

Stock Number 1—MS Fe EDTA 100× Stock contains (per liter): 3.724 g ofNa₂ EDTA (Add first, dissolve in dark bottle while stirring); and 2.784g of FeSO₄ —7H₂O.

Stock Number 2—MS Sulfate 100× stock contains (per liter): 37.0 g ofMgSO₄-7H₂O; 1.69 g of MnSO₄—H₂O; 0.86 g of ZnSO₄-7H₂O; and 0.0025 g ofCuSO₄-5H₂O.

Stock Number 3-FN Lite Halides 100× Stock contains (per liter): 30.0 gof CaCl₂-2H₂O; 0.083 g of KI; and 0.0025 g of CoCl₂-6H₂O.

Stock Number 4-FN Lite P, B, Mo 100× Stock contains (per liter): 18.5 gof KH₂PO₄; 0.62 g of H₃BO₃; and 0.025 g of Na₂MoO₄-2H₂O.

SB1 Solid Medium contains the following (per liter): 1 package MS salts(Gibco/BRL-Cat. No. 11117-066); 1 mL B5 vitamins 1000× stock; 31.5 gGlucose; 2 mL 2,4-D (20 mg/L final concentration); adjust to pH 5.7; and8 g TC agar.

SB199 Solid Medium contains the following (per liter): 1 package MSsalts (Gibco/BRL-Cat. No. 11117-066); 1 mL B5 vitamins 1000× stock; 30 gSucrose; 4 mL 2,4-D (40 mg/L final concentration); adjust to pH 7.0; and2 gm GELRITE®.

SB166 Solid Medium contains the following (per liter): 1 package MSsalts (Gibco/BRL-Cat. No. 11117-066); 1 mL B5 vitamins 1000× stock; 60 gmaltose; 750 mg MgCl₂ hexahydrate; 5 g Activated charcoal; adjust to pH5.7; and 2 g GELRITE®.

SB103 Solid Medium contains the following (per liter): 1 package MSsalts (Gibco/BRL-Cat. No. 11117-066); 1 mL B5 vitamins 1000× stock; 60 gmaltose; 750 mg MgCl2 hexahydrate; adjust to pH 5.7; and 2 g GELRITE®.

SB 71-4 Solid Medium contains the following (per liter): 1 bottleGamborg's B5 salts w/ sucrose (Gibco/BRL-Cat. No. 21153-036); adjust topH 5.7; and 5 g TC agar.

2,4-D Stock: Obtain Premade From PHYTOTECHNOLOGY LABORATORIES™ Cat. No.D 295—concentration 1 mg/mL.

B5 Vitamins Stock contains the following (per 100 mL): 10 gMyo-inositol; 100 mg Nicotinic acid; 100 mg Pyridoxine HCl; and 1 gThiamine. If the solution does not dissolve quickly enough, apply a lowlevel of heat via the hot stir plate. Store aliquots at −20° C.

SB 228—Soybean Histodifferentiation & Maturation (SHaM) contains thefollowing (per liter): 600 mL DDI H₂O; 100 mL FN-Lite Macro Salts forSHaM 10×; 1 mL MS Micro Salts 1000×; 10 mL MS FeEDTA 100×; 6.82 mL CaCl100×; 1 mL B5 Vitamins 1000×; 0.149 g L-Methionine; 30 g Sucrose; 30 gSorbitol; adjust volume to 900 mL; adjust to pH 5.8; autoclave. Add tocooled media (<30° C.): 110 mL 4% glutamine (final conc. 30 mM). Finalvolume will be 1010 mL after glutamine addition. Because glutaminedegrades relatively rapidly, it may be preferable to add immediatelyprior to using media. Expiration is 2 weeks after glutamine is added;base media can be kept longer without glutamine.

FN-lite Macro for SHAM 10×-Stock #1 contains the following (per liter):4.63 g (NH₄)2SO₄ (Ammonium Sulfate); 28.3 g KNO₃ (Potassium Nitrate);3.7 g MgSO₄*7H₂O (Magnesium Sulfate Heptahydrate); 1.85 g KH₂PO₄(Potassium Phosphate, Monobasic); bring to volume; autoclave.

MS Micro 1000-Stock #2 contains the following (per 1 liter): 6.2 g H₃BO₃(Boric Acid); 16.9 g MnSO₄*H₂O (Manganese Sulfate Monohydrate); 8.6 gZnSO4*7H20 (Zinc Sulfate Heptahydrate); 0.25 g Na₂MoO₄*2H20 (SodiumMolybdate Dihydrate); 0.025 g CuSO₄*5H₂0 (Copper Sulfate Pentahydrate);0.025 g CoCl₂*6H₂0 (Cobalt Chloride Hexahydrate); 0.8300 g KI (PotassiumIodide); bring to volume and autoclave.

FeEDTA 100×-Stock #3 contains the following (per liter): 3.73 g Na₂EDTA(Sodium EDTA); EDTA must be completely dissolved before adding iron;2.78 g FeSO₄*7H₂0 (Iron Sulfate Heptahydrate); bring to volume andautoclave. Solution is photosensitive. Bottle(s) should be wrapped infoil to omit light.

Ca 100×-Stock #4 contains the following (per liter): 44 g CaCl₂*2H₂0(Calcium Chloride Dihydrate); bring to volume and autoclave.

B5 Vitamin 1000×-Stock #5 contains the following (per liter): 10 gThiamine*HCl; 1 g Nicotinic Acid; 1 g Pyridoxine*HCl; 100 gMyo-Inositol; bring to volume; store frozen.

4% Glutamine—Stock #6 contains the following (per liter): 900 mL DDIwater heated to 30° C.; 40 g L-Glutamine; gradually add while stirringand applying low heat. Do not exceed 35° C. Bring to Volume. Filterterilize and store frozen. Warm thawed stock in 31° C. bath to fullydissolve crystals.

Tocol and Oil Analysis:

Somatic embryos were harvested after two weeks of culture in the liquidmaturation medium SB228 (SHaM) liquid media. Thirty-one events werecreated. All embryos generated for a given event were harvested in bulkand processed as follows. Embryos were frozen on dry ice or byincubation in a −80° C. freezer for two h followed by lyophilization for48 h.

Dried embryos were ground to a fine powder using a genogrinder vial(½″×2″ polycarbonate) and a steel ball (SPEX Centriprep (Metuchen, N.J.,U.S.A.). Grinding time was 30 sec at 1450 oscillations per min. Forevery event, approximately 30 mg of tissue were weighed into Eppendorftubes 5 μL of tocopherol acteate (3.79 ng μL⁻¹) was added to each sampleas internal standard. The tissue was extracted using 200 μL heptane atroom temperature under continuous shaking for 2 h. Heptane extracts werecleared by centrifugation and filtration and 10 uL of extract wereanalyzed by HPLC as described in Example 3.

Tocol data are summarized in Table 19.

TABLE 19 Tocol Composition (percent of total tocopherols (tocph.) andtocotrienols (toct.)) of Soybean Somatic Embryos Generated byCo-Transformation with KS325xSC38 and pKR561-MCVTE3 Event alpha- beta-gamma- delta- alpha- beta- gamma- delta- tocph. toct. ID tocoph. tocoph.tocoph. tocoph. toct. toct. toct. toct. (ppm) (ppm) 19 17 1 0 0 78 3 1 0247 1123 11 19 0 0 0 77 2 1 0 317 1328 28 27 3 0 0 57 11 1 0 161 370 2930 6 1 0 47 16 1 0 262 454 1 31 7 0 0 42 18 1 1 302 497 7 17 7 8 1 35 252 6 199 411 23 32 3 8 1 34 10 10 3 244 326 31 18 3 3 1 34 14 18 9 201586 2 56 2 2 0 32 7 1 0 284 190 12 24 3 29 2 25 8 5 3 225 158 30 18 4 242 24 13 8 7 184 204 17 5 0 16 0 7 0 70 1 182 669 15 13 1 12 4 6 2 42 20186 427 14 15 1 66 6 4 0 5 1 241 30 8 5 1 12 5 4 2 50 21 188 642 5 7 115 4 3 0 51 20 189 508 4 6 1 13 4 2 0 54 19 224 692 13 2 1 6 5 2 0 45 39148 941 27 4 0 13 0 2 0 80 1 175 832 20 6 0 15 5 1 0 37 35 171 472 25 521 6 2 1 0 23 15 255 162 9 5 0 38 3 1 0 43 9 210 241 32 6 0 88 4 1 0 1 0165 3 26 7 0 89 3 0 0 1 0 222 2 18 5 0 89 4 0 0 1 0 300 6 16 6 0 80 4 08 1 0 287 32 22 11 0 80 6 0 0 2 0 238 7 10 9 0 84 5 0 0 1 0 266 4 21 1 09 4 0 0 54 32 165 1002 3 8 1 86 4 0 0 1 0 225 3 6 66 5 27 2 0 0 1 0 2561 24 98 2 0 0 0 0 0 0 361 0

Oil concentration of the heptane extract was measured as follows. 25 μLof extract was derivatized to fatty acid methyl esters as follows. OnemL of a 25% sodium methoxide stock solution was added to 24 mL of HPLCgrade methanol. Sodium methoxide was stored under an inert gas.

Five μL of a 17:0 TAG (Nu-Chek Prep, Elysian, Minn., USA) stock solution(10 mg/mL) was combined with 25 μL of heptane tissue extract in a glassculture tube 500 μL of 1% sodium methoxide was added. Samples werederivatized in a water bath at 50° C. for 15 min. Samples were allowedto cool to RT and 1 mL of 1M NaCl was added followed by brief mixing.FAMEs were extracted into 1 mL of heptane and 4 μL sample werequantitated by GC analysis.

Two transgenic somatic embryo events (with Event ID numbers 19 and 11)were identified that contained very high levels of alpha tocotrienol(>70% of total tocols). These events contained 1270 and 1060 ppm alphatocotrienol on a DW basis and 21,590 and 18050 ppm alpha tocotrienol onan oil basis. Co-expression of HV HGGT, ZM GTMT and MC VTE3 allowed forvery high level of alpha tocotrienol accumulation. A vitamin E profilewas generated that was dominated by alpha tocotrienol and alphatocopherol; other vitamins represented less than 5% of the total tocols.Moreover, the tocol profile of a significant number of somatic embryoevents suggest that only some of the genes present on the two DNAfragments used for transformation were expressed in these. For example,event number 24 very likely only expressed ZM GTMT and MC VTE3 andevents 19 and 21 only expressed HV HGGT (see Example 3). Event number 7very likely only expressed HGGT and ZM GTMT (see Example 7). Its tocolprofile is very similar to that of soybeans expressing only these twovitamin E biosynthetic genes. Events 27 and 17 with a profile dominatedby gamma tocotrienol very likely only expressed HV HGGT and MC VTE3.Finally, events such as 32, 26, 18 and 10, with a tocol profiledominated by gamma tocopherol and only trace levels of tocotrienol, verylikely did not express any transgene-derived Vitamin E biosyntheticgenes.

In summary, the data illustrated that by using only three types ofvitamin E biosynthetic genes described herein, a wide range of vitamin Eprofiles can be generated in a combinatorial fashion. Moreover, it wasshown that co-expression of HV-HGGT, ZM GTMT (VTE4) and MC VTE3 can leadto an increase of tocol levels of 6.5-fold and an increase of therelative alpha tocochromanol content to >95% of total tocols.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication or patent application was specifically andindividually indicated to be incorporated by reference in its entirety.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of ordinary skill will recognize that certain changesand modifications may be practiced and are included within the scope ofthe foregoing invention and the appended claims.

What is claimed is:
 1. A method of increasing the level of alphatocotrienol, beta-tocotrienol, or both, in a microbial cell, comprising:stably incorporating into the genome of a microbial cell: (a) a firstrecombinant nucleic acid molecule comprising at least one regulatorysequence operably linked to at least one nucleotide sequence selectedfrom the group consisting of: (i) the nucleotide sequence set forth inSEQ ID NO:15; (ii) a nucleotide sequence encoding the amino acidsequence set forth in SEQ ID NO:16; and (iii) a nucleotide sequenceencoding a polypeptide having an amino acid sequence of at least 95%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:16, wherein the polypeptide has gammatocopherolmethyltransferase activity; (b) a second recombinant nucleic acidmolecule comprising at least one regulatory sequence operably linked toat least one nucleotide sequence selected from the group consisting of:(iv) the nucleotide sequence set forth in SEQ ID NO:1; (v) a nucleotidesequence encoding the amino acid sequence set forth in SEQ ID NO:2; and(vi) a nucleotide sequence encoding a polypeptide having an amino acidsequence of at least 95% sequence identity, based on the Clustal Vmethod of alignment, when compared to SEQ ID NO:2, wherein thepolypeptide has homogentisate geranylgeranyl transferase activity; and(c) a third recombinant nucleic acid molecule comprising at least oneregulatory sequence operably linked to at least one nucleotide sequenceselected from the group consisting of: (vii) the nucleotide sequence setforth in SEQ ID NO:53; (viii) a nucleotide sequence encoding the aminoacid sequence set forth in SEQ ID NO:54; and (ix) a nucleotide sequenceencoding a polypeptide having an amino acid sequence of at least 95%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:54, wherein the polypeptide has 2-methyl-6-phytylbenzoquinol methyltransferase activity; and selecting atransformed microbial cell that expresses the nucleotide sequence of thefirst recombinant nucleic acid molecule, the nucleotide sequence of thesecond recombinant nucleic acid molecule, and the nucleotide sequence ofthe third recombinant molecule wherein said transformed microbial cellhas an increased level of alpha-tocotrienol, beta-tocotrienol, or both,relative to a microbial cell with a similar genetic background butlacking said first recombinant nucleic acid molecule, said secondrecombinant nucleic acid molecule, and said third recombinant nucleicacid molecule.
 2. The method of claim 1, wherein said first, second andthird recombinant nucleic acid molecules are incorporated into thegenome of a microbial cell by co-transformation.
 3. The method of claim1, wherein at least one of said first, second or third recombinantnucleic acid molecules is incorporated into the genome of a microbialcell by re-transformation of a transformed microbial cell, wherein saidtransformed microbial cell comprises at least one of said first, secondor third recombinant nucleic acid molecules.
 4. The method of claim 1,wherein said microbial cell is an algal cell.
 5. The method of claim 1,wherein the at least one regulatory sequence of said first, said second,and said third recombinant nucleic acid molecules is a promoter.